Secondary Logo

Transgenic overexpression of glutathione S-transferase μ-type 1 reduces hypertension and oxidative stress in the stroke-prone spontaneously hypertensive rat

Olson, Erina; Pravenec, Michalb; Landa, Vladimirb; Koh-Tan, H.H. Calinea; Dominiczak, Anna F.a; McBride, Martin W.a,*; Graham, Delytha,*

doi: 10.1097/HJH.0000000000001960
ORIGINAL PAPERS: Organ damage/mechanistic aspects
Open
SDC

Background: Combined congenic breeding and microarray gene expression profiling previously identified glutathione S-transferase μ-type 1 (Gstm1) as a positional and functional candidate gene for blood pressure (BP) regulation in the stroke-prone spontaneously hypertensive (SHRSP) rat. Renal Gstm1 expression in SHRSP rats is significantly reduced when compared with normotensive Wistar Kyoto (WKY) rats. As Gstm1 plays an important role in the secondary defence against oxidative stress, significantly lower expression levels may be functionally relevant in the development of hypertension. The aim of this study was to investigate the role of Gstm1 in BP regulation and oxidative stress by transgenic overexpression of the Gstm1 gene.

Method: Two independent Gstm1 transgenic SHRSP lines were generated by microinjecting SHRSP embryos with a linear construct controlled by the EF-1α promoter encoding WKY Gstm1 cDNA [SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY].

Results: Transgenic rats exhibit significantly reduced BP and pulse pressure when compared with SHRSP [systolic: SHRSP 205.2 ± 3.7 mmHg vs. SHRSP-Tg(Gstm1)1WKY 175.5 ± 1.6 mmHg and SHRSP-Tg(Gstm1)2WKY 172 ± 3.2 mmHg, P < 0.001; pulse pressure: SHRSP 58.4 ± 0.73 mmHg vs. SHRSP-Tg(Gstm1)1WKY 52.7 ± 0.19 mmHg and SHRSP-Tg(Gstm1)2WKY 40.7 ± 0.53 mmHg, P < 0.001]. Total renal and aortic Gstm1 expression in transgenic animals was significantly increased compared with SHRSP [renal relative quantification (RQ): SHRSP-Tg(Gstm1)1WKY 1.95 vs. SHRSP 1.0, P < 0.01; aorta RQ: SHRSP-Tg(Gstm1)1WKY 2.8 vs. SHRSP 1.0, P < 0.05]. Renal lipid peroxidation (malondialdehyde: protein) and oxidized : reduced glutathione ratio levels were significantly reduced in both transgenic lines when compared with SHRSP [malondialdehyde: SHRSP 0.04 ± 0.009 μmol/l vs. SHRSP-Tg(Gstm1)1WKY 0.024 ± 0.002 μmol/l and SHRSP-Tg(Gstm1)2WKY 0.021 ± 0.002 μmol/l; (oxidized : reduced glutathione ratio): SHRSP 5.19 ± 2.26 μmol/l vs. SHRSP-Tg(Gstm1)1WKY 0.17 ± 0.11 μmol/l and SHRSP-Tg(Gstm1)2WKY 0.47 ± 0.22 μmol/l]. Transgenic SHRSP rats containing the WKY Gstm1 gene demonstrate significantly lower BP, reduced oxidative stress and improved levels of renal Gstm1 expression.

Conclusion: These data support the hypothesis that reduced renal Gstm1 plays a role in the development of hypertension.

aInstitute of Cardiovascular & Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK

bInstitute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic

Correspondence to Delyth Graham, Dr, Institute of Cardiovascular & Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK. Tel: +44 0 141 3302524; fax: +44 0 141 3306997; e-mail: Delyth.Graham@glasgow.ac.uk

Abbreviations: BP, blood pressure; GSSG : GSH, oxidized: reduced glutathione ratio; GSTM1, glutathione S-transferase μ-type 1; LVMI, left ventricular mass index/indices; PP, pulse pressure; QTL, quantitative trait locus/loci; RMI, renal mass index/indices; ROS, reactive oxygen species; SHRSP, stroke-prone spontaneously hypertensive rat; WKY, Wistar Kyoto

Received 4 March, 2018

Accepted 10 September, 2018

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.jhypertension.com).

This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/4.0

Back to Top | Article Outline

INTRODUCTION

Human essential hypertension is a complex polygenic disease with genetic heritability averaging approximately 40% and with strong influence of environmental factors and gene–environment interaction [1,2]. Heterogeneity in the general population and the polygenic complexities of the disease has meant that identification and functional validation of candidate genes has proved difficult in humans. A range of studies including genome wide association studies, meta-analysis and admixture mapping studies have successfully identified numerous loci associated with phenotypic variance for SBP and DBP [2–15]. However, only a small portion of the variance of blood pressure (BP) (i.e. ∼5%) is explained by the loci discovered so far. To fully understand the functional roles of genetic loci on BP regulation it is essential to interrogate their impact in suitable rodent models. Rodent models are commonly used to discern and dissect genetic determinants of hypertension as they offer more favourable investigative opportunities because of reduced genetic heterogeneity, the capacity for controlled breeding and environmental conditions, and the ability to produce genetic crosses and analyse large numbers of progeny [16,17]. This translational approach will improve our knowledge and understanding of pathways, networks and gene environment interaction underlying essential hypertension.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a well characterized experimental model for human essential hypertension, which develops a number of cardiovascular complications, including cardiac hypertrophy and stroke [18–20]. Similar to human disease, the genetic determination of BP variation in this model is complex and the result of multiple gene–gene and gene–environment interactions [21,22]. Genome-wide linkage studies have proved successful in the localization of large chromosomal regions containing quantitative trait loci (QTLs) for blood regulation in the SHRSP. In particular, previous work in our laboratory has identified at least two BP QTLs mapping to rat chromosome 2 [20]. These QTL were subsequently confirmed with the production and phenotypic analysis of chromosome 2 congenic strains, generated by introgressing regions of rat chromosome 2 from the normotensive Wistar Kyoto (WKY) strain into the SHRSP genetic background. This resulted in significant reduction of SBP and DBP when the congenic strains were compared with the SHRSP parental strain [23,24].

Combining this congenic strategy with genome-wide microarray expression profiling allowed the identification of the positional candidate gene, glutathione S-transferase μ-type 1 (Gstm1) [24,25]. This gene encodes a cytoplasmic glutathione S-transferase (GST) that belongs to the mu class of GST enzymes, which function in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione [26]. GSTM1 protein is expressed in a wide range of tissues including the liver, endocrine tissues, brain, muscle tissues and kidney [27]. Our previous studies in the SHRSP rat demonstrate significantly downregulated expression of Gstm1 in the kidney when compared with the WKY. As a result of this reduced Gstm1 expression we hypothesise that the secondary defence against oxidative stress is compromised in SHRSP rats leading to oxidative stress-induced hypertension and end organ damage.

The aim of this study was to establish conclusive evidence that reduced Gstm1 expression affects BP regulation and oxidative stress. Two independent SHRSP transgenic lines were created with the aim of reversing Gstm1 deficiency by incorporation of the Gstm1 gene from the normotensive WKY strain into the SHRSP genome.

Back to Top | Article Outline

MATERIAL AND METHODS

Animal strains

Inbred colonies of SHRSP and WKY have been maintained at the University of Glasgow since 1991, as described previously [20]. All animals were housed under controlled environmental conditions, fed standard rat chow (rat and mouse no. 1 maintenance diet, Special Diet Services) and water provided ad libitum. All animal procedures performed were approved by the Home Office according to regulations regarding experiments with animals in the United Kingdom.

Back to Top | Article Outline

Transgenic rat generation

Two independent transgenic lines of Gstm1 SHRSP rats were created through incorporation of the Gstm1 gene from the normotensive WKY strain into the SHRSP genome, which are designated SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY. Generation of these transgenic SHRSP rats involved microinjection of a 2.7-kb linear construct encoding wild-type (WKY) Gstm1 under the control of the universal EF-1α promoter (Supplementary Fig. 1, http://links.lww.com/HJH/B27). They were generated using the same expression platform and microinjection fragment purification protocol employed as previously described [28]. See Supplementary file for extended methods.

Back to Top | Article Outline

Phenotypic measurements

The Dataquest IV telemetry system (Data Sciences International) was used for the direct measurement of SBP, DBP, pulse pressure (PP), activity and heart rate (HR) [23,29]. Male rats were implanted at 12 weeks of age with 1-week recovery, and haemodynamic parameters recorded until rats were 21 weeks of age. BP prior to 12 weeks of age was measured by tail cuff plethysmography [30]. Metabolic cages were used to collect 24-h urine samples from all animals before sacrifice. At sacrifice, weights for cardiac mass index, left ventricular (LV) mass index (LVMI) and renal mass index (RMI) were measured and corrected to tibia length. At sacrifice a range of tissues were harvested and either snap frozen in liquid nitrogen and stored at −80 °C or fixed in 4% buffered formaldehyde and paraffin embedded for histology or immunohistochemistry (IHC).

Urinary protein was measured using Thermo Pierce Protein Assay 660 (no. 22662; Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) with samples diluted one in five for assay. Urinary biochemistry analysis was carried out using a Beckman Coulter AU640 clinical chemistry analyser utilizing ion selective electrodes. Indirect glomerular filtration rates (GFR) were determined by a clinically validated automated analyser (c311, Roche Diagnostics, Burgess Hill, UK), using the manufacturer's calibrators and quality control material for isotope dilution-mass spectrometry. All measurements were normalized to kidney weight.

Back to Top | Article Outline

Echocardiography

Transthoracic echocardiography was used to assess cardiac geometry and contractility, as previously described [30,31]. Echocardiography was performed prior to sacrifice at 21 weeks of age. Animals were sedated and short axis 2-dimensional B-mode and M-mode images were taken through the left parasternal window at the papillary muscle levels using an ACUSON Sequoia C512 Echocardiograph (Siemens, Erlangen, Germany). Averaged data from six consecutive cardiac cycles from each M-mode tracing were used in the following equation for the calculation of LV mass [American society of echocardiography (ASE)-cube formula with Devereux correction factor] [LV mass = 0.8 (1.04 ((EDD + PWT + AWT) 3 − EDD3)) + 0.6], where PWT is the posterior wall thickness (mm), AWT is the anterior wall thickness (mm), EDD is the end diastolic dimension (mm). LV end-systolic volume (ESV) and LV end-diastolic volume (EDV) can be calculated from two-dimensional images according to a modified Simpson's rule. LV ejection fraction (LVEF) is then determined from EDV and ESV. Cardiac index is estimated as cardiac output (CO) adjusted for tibia length.

Back to Top | Article Outline

Renal histology

To assess evidence of renal disease, 3 μm sections from WKY, SHRSP and transgenic rat kidneys were stained with Harris haematoxylin and eosin and examined using an Olympus DP72 camera attached to Olympus BX51 microscope, and analysed using DP2-BSW software (Olympus, Hamburg, Germany).

Back to Top | Article Outline

Quantitative real-time PCR

Total RNA was extracted from kidney, aorta, heart and brain tissues from 5-week-old and 21-week-old rats using RNeasy kits (Thermo Fisher Scientific Inc.), treated with DNase Free (Thermo Fisher Scientific Inc.) and quantified using Ribogreen (Thermo Fisher Scientific Inc.). Normalization was confirmed by performing real-time PCR on TaqMan (Thermo Fisher Scientific Inc.) of β-actin with comparable threshold cycles. TaqMan probes for Gstm1 (Rn00755117m1-labeled FAM) and β-actin (4352340E-labeled VIC) were multiplexed. Expression of Gstm1 relative to β-actin in each sample was derived using the comparative (ΔΔCT) method [25].

For transgene or WKY (wild-type) gene expression, Exiqon custom locked-nucleic acid SYBR Green probes were designed to single nucleotide polymorphism (SNP) differences between WKY and SHRSP in exon 8 as previously described [25].

Back to Top | Article Outline

Western analysis of glutathione S-transferase μ-type 1 in rat kidney

Kidneys from 5-week-old and 21-week-old SHRSP, WKY and SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats were homogenized in protease inhibitor-containing buffer. Protein concentration was determined using a Bio-Rad BCA kit. Proteins were separated by PAGE and electroblotted onto a Hybond-P membrane (Thermo Fisher Scientific Inc,). Membranes were incubated with the appropriate primary antibody (anti-GSTM1 [25] 1 : 5000, antiβ-actin 1 : 1000) before repeated washing and application of a horseradish peroxidase-conjugated secondary antibody. Protein bands were detected by chemiluminescence (ECL kit; Thermo Fisher Scientific Inc), and visualized and quantified using a Bio-Rad Image Analyzer densitometry system.

Back to Top | Article Outline

Immunohistochemistry

Sections (6 μm) were obtained from kidneys and aorta of SHRSP, WKY, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY male rats. Sections were blocked for 60 min with 2% serum in PBS, followed by overnight incubation at 26 °C with an antibody against GSTM1 [25]. A biotinylated secondary antibody, diluted in blocking reagent (ABC universal kit, two drops blocking serum + two drops supplied vectastain biotinylated antibody + 5 ml PBS) was added for 30 min at room temperature (RT). 3,3’-Diaminobenzidine (DAB) chromogen (DAB substrate kit) for universal and peroxidase secondary antibody was prepared following manufacturer's instructions. After treatment with antigen retrieval solution (Agilent, Stockport, Cheshire, UK), sections were blocked with 20% serum in PBS for 1 h. Washing with PBS was followed by incubation with a secondary antibody, at RT for 1 h. Sections were counterstained with Haematoxylin. Images shown were taken with Olympus DP72 attached to Olympus BX51 microscope, using DP2-BSW software (Olympus, Hamburg, Germany).

Back to Top | Article Outline

Oxidative stress measurements

Superoxide, hydrogen peroxide, nitric oxide, glutathione and lipid peroxidation measurements were performed in homogenized snap-frozen tissues from 5-week-old and 21-week-old animals. Superoxide was measured using lucigenin chemiluminescence in kidney tissue homogenized in lysis buffer [20 mmol/l of KH2PO4, 1 mmol/l of ethylene glycol-bis(B-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 1 μg/ml of aprotinin, 1 μg/ml of leupeptin, 1 μg/ml of pepstatin and 1 mmol/l of phenylmethylsulfonyl fluoride (PMSF)]. Fifty microliters of the sample were added to a suspension containing 175 μl of assay buffer (50 mmol/l of KH2PO4, 1 mmol/l of EGTA, and 150 mmol/l of sucrose) and lucigenin (5 μmol/l). NADPH (10−4 mol/l) was added to the suspension (300 μl) containing lucigenin. Luminescence was measured every 18 s for 3 min by a luminometer (AutoLumat LB 953; Berthold Technologies, Wildbad, Germany) before and after stimulation with NADPH. A buffer blank was subtracted from each reading. The results are expressed as counts per milligram of protein (percentage of control).

Glutathione levels were measured according to Cayman Glutathione Assay Kit (#703002) manufacturer's instructions. Hydrogen peroxide levels were measured using Invitrogen's AmplexR Red Hydrogen Peroxide/Peroxidase Assay Kit Amplex Red (Thermo Fisher Scientific Inc.) for tissues. Total nitrate and nitrite concentrations were measured using the Cayman Biochemical Nitrate/Nitrite Colorimetric Assay Kit.

Back to Top | Article Outline

Statistical analysis

Results are expressed as mean ± SEM. Haemodynamic parameters for WKY or transgenic strains were compared with SHRSP, using repeated measures analysis of variance (ANOVA) (general linear model). Other phenotypic comparisons between groups were performed by one way ANOVA with Tukey's multiple comparison test, unless stated otherwise. For cardiac parameter analysis statistical analysis was performed by a Student's t test.

Back to Top | Article Outline

RESULTS

Haemodynamic parameters

When measured by radiotelemetry, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats demonstrated significantly reduced SBP (Fig. 1a), and PP (Fig. 1c) when compared with SHRSP (P < 0.001). DBP (Fig. 1b) and mean arterial pressure (Fig. 1d) were significantly reduced across the entire analysis period in SHRSP-Tg(Gstm1)1WKY rats, whereas SHRSP-Tg(Gstm1)2WKY rats demonstrated a significant reduction in diastolic and mean arterial pressure from approximately 18 weeks of age onwards (P < 0.001). There were no significant differences in HR or activity level across the measurement period between the four strains (Supplementary Fig. 2, http://links.lww.com/HJH/B27). Before 12 weeks of age, SBP measured by tail cuff plethysmography was significantly lower in WKY and the transgenic lines when compared with SHRSP (P < 0.001) (Supplementary Table 1, http://links.lww.com/HJH/B27).

FIGURE 1

FIGURE 1

Back to Top | Article Outline

Cardiac parameters

Cardiac parameters were assessed in rats at 21 weeks of age by transthoracic echocardiography immediately prior to sacrifice. LVMI was significantly reduced in WKY and SHRSP-Tg(Gstm1)2WKY hearts compared with that of the SHRSP (P < 0.05). SHRSP-Tg(Gstm1)1WKY rats displayed a trend towards a reduction in LVMI but did not reach statistical significance (Fig. 2a). Relative wall thickness in SHRSP-Tg(Gstm1)1WKY, SHRSP-Tg(Gstm1)2WKY and WKY rats was significantly lower compared with that of the SHRSP (F = 7.9, P < 0.05) (Fig. 2b). Stroke volume (SV) for SHRSP rats was significantly decreased when compared with WKY, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats (Fig. 2c). CO in SHRSP rats was significantly decreased when compared with WKY, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats (Fig. 2d). There were no significant differences in LV fractional shortening or ejection fraction between WKY, SHRSP, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY (data not shown).

FIGURE 2

FIGURE 2

Back to Top | Article Outline

Renal parameters

Renal function data at 21 weeks of age are given in Table 1. Although there was a trend towards increased estimated GFR in SHRSP-Tg(Gstm1)1WKY rats, there was no significance difference between all four strains (P > 0.05). There was also no significant difference between all four strains for urine sodium, potassium and chloride levels (P > 0.05). Levels of proteinuria in WKY, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats were significantly lower than that of the SHRSP (P < 0.05). At 21 weeks of age, kidney mass, normalized to body weight, was significantly lower in WKY when compared with SHRSP (P < 0.0001). RMI for SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats was not significantly different when compared with SHRSP.

TABLE 1

TABLE 1

Renal morphology, assessed by haemotoxylin and eosin staining at 21 weeks of age, showed normal arterioles in the WKY and transgenic animals, however renal arterioles in the SHRSP showed evidence of hyperplasia, a sign of accelerated hypertension (Fig. 3).

FIGURE 3

FIGURE 3

Back to Top | Article Outline

Renal glutathione S-transferase μ-type 1 mRNA and protein expression

Total Gstm1 mRNA expression (Fig. 4a) and transgene specific (WKY variant of Gstm1) mRNA expression (Fig. 4b) in kidneys from rats at 5 weeks of age was significantly higher in SHRSP-Tg(Gstm1)1WKY, SHRSP-Tg(Gstm1)2WKY and WKY rats compared with SHRSP (P < 0.05). This increase in renal Gstm1 mRNA expression was paralleled by GSTM1 protein levels in SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats. Western blot analysis indicated significantly increased GSTM1 protein expression in SHRSP-Tg(Gstm1)2WKY and WKY when compared with SHRSP, with a trend towards an increase in kidneys from SHRSP-Tg(Gstm1)1WKY rats (Fig. 4c and d). IHC in kidneys from 5-week-old rats confirmed increased protein expression within the distal tubules of WKY and SHRSP-Tg(Gstm1)1WKY rats when compared with SHRSP (Fig. 4e).

FIGURE 4

FIGURE 4

At 21 weeks of age, total renal Gstm1 mRNA expression (Fig. 5a) and transgene specific mRNA expression (Fig. 5b) were significantly higher in WKY rats when compared with with SHRSP (P < 0.05). Transgene specific mRNA expression was also significantly increased in SHRSP-Tg(Gstm1)1WKY rats, however SHRSP-Tg(Gstm1)2WKY rats showed low transgene expression, which was not different from SHRSP (Fig. 5b). GSTM1 protein levels were significantly higher in WKY rats compared with that of the SHRSP (P < 0.01) but were not significantly different in SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats compared with SHRSP (P > 0.05) (Fig. 5c and d). IHC confirmed an increase in GSTM1 protein expression in distal tubules of WKY rats compared with SHRSP, with no increase in GSTM1 protein expression in the transgenic rats (Fig. 5e).

FIGURE 5

FIGURE 5

Back to Top | Article Outline

Vascular glutathione S-transferase μ-type 1 mRNA expression and immunohistochemistry

At 5 weeks of age vascular (aortic) total Gstm1 expression showed a trend towards an increase in SHRSP-Tg(Gstm1)1WKY and WKY rats, and was significantly increased in SHRSP-Tg(Gstm1)2WKY rats when compared with the SHRSP (P < 0.05) (Supplementary Fig. 3A, http://links.lww.com/HJH/B27). At 21 weeks of age, Gstm1 mRNA expression in aorta was significantly increased in SHRSP-Tg(Gstm1)1WKY and WKY rats when compared with and SHRSP (P < 0.005) (Supplementary Fig. 3B, http://links.lww.com/HJH/B27). However, aortic Gstm1 expression in SHRSP-Tg(Gstm1)2WKY rats showed an increased trend but was not significantly different from WKY, SHRSP-Tg(Gstm1)1WKY or SHRSP rats. IHC was performed on the aorta at 21 weeks of age to investigate GSTM1 protein expression. When quantified, percentage staining in the aorta demonstrated a significant increase in GSTM1 protein in WKY rats when compared with SHRSP (P < 0.05) (Supplementary Fig. 3C and D, http://links.lww.com/HJH/B27). There was a trend towards an increase in protein expression for aortas in SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats which was not significantly different from either parental strain, indicating intermediate expression (Supplementary Fig. 3D, http://links.lww.com/HJH/B27).

Back to Top | Article Outline

Cardiac glutathione S-transferase μ-type 1 mRNA expression

At 21 weeks of age, cardiac total Gstm1 expression was significantly increased in the SHRSP-Tg(Gstm1)1WKY line when compared with WKY and SHRSP (P < 0.05). Cardiac Gstm1 expression in SHRSP-Tg(Gstm1)2WKY rats showed a trend towards increase, but was not significantly different from WKY, SHRSP-Tg(Gstm1)1WKY or SHRSP rats (Supplementary Fig. 4A, http://links.lww.com/HJH/B27).

Back to Top | Article Outline

Effects of glutathione S-transferase μ-type 1 expression on renal and cardiac oxidative stress

At 21 weeks of age there was no significant difference between SHRSP, WKY or the transgenic rat lines for superoxide (O2) levels in whole kidney homogenates (Fig. 6a), as measured by lucigenin chemiluminesence, or for hydrogen peroxide (Fig. 6b), as measured by Amplex Red Assay (P > 0.05). Oxidized : reduced glutathione (GSSG : GSH) ratios, showed a trend towards reduced levels in the WKY and both transgenic lines when compared with the SHRSP at 21 weeks of age (Fig. 6c). Lipid peroxidation measured using malondialdehyde (MDA) assay showed a significant reduction in renal MDA production in the WKY, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY when compared with the SHRSP (P < 0.05), Fig. 6d. There were no significant differences in renal GSSG : GSH ratios between the four strains at 5 weeks of age (Supplementary Table 2, http://links.lww.com/HJH/B27), however renal GSSG : GSH ratios were significantly lower at 5 weeks of age when compared with 21 weeks of age (Supplementary Table 2, http://links.lww.com/HJH/B27).

FIGURE 6

FIGURE 6

Back to Top | Article Outline

DISCUSSION

In this study, the investigation of two independently generated transgenic rat lines, SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY, has demonstrated that increased expression of Gstm1 improves BP regulation and reduces oxidative stress in the SHRSP rat. Gstm1 deficiency was reversed by incorporation of the WKY Gstm1 gene into the SHRSP genome leading to a significant reduction in SBP, DPB, MAP and PP. These haemodynamic improvements were paralleled by significant reductions in cardiac hypertrophy and improved cardiac function in both transgenic lines. Furthermore, renal oxidative stress, indicated by lipid peroxidation and GSSG : GSH ratio, was significantly reduced in both transgenic lines when compared with SHRSP. These findings support the hypothesis that reduced Gstm1 expression plays a causal role in the development of oxidative stress and BP elevation in the SHRSP rat.

The method of random transgene integration employed in this study may be viewed as a limitation when compared with the locus specific genome editing afforded by clustered regularly interspaced short palindromic repeats (CRISPR) technology [32]. However, this method for gene overexpression is still regularly used to generate genetically engineered rodent models, requires short development time and importantly is a method that we have previous success with in rats [33–37]. The generation of more than one transgenic line, using the same transgene and promoter, is critical to confirm that the phenotypic differences are the result of the transgene itself and not due to positional effects caused at the random insertion site. In independently generated lines it is highly unlikely that the transgene will be inserted into the identical genomic position. If significant phenotypic changes of similar magnitude and direction are observed for two independently generated lines then this provides corroborative evidence that the phenotype differences are due to a functional effect of the transgene.

Although both SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY transgenic rats demonstrate improved cardiovascular profiles, each line showed some unique molecular and phenotypic expression patterns. For example, while DBP for both SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY lines is significantly lower than that of SHRSP, the diastolic pressure profiles differ in the rate of increase over time (Fig. 1b). Similar phenotypic diversity between two independently generated lines has been demonstrated in the transgenic rescue of CD36 in the SHR rat, whereby, unique differences in insulin resistance and BP regulation were observed between two transgenic lines despite insertion of the same CD36 transgene and promoter [28]. It is clear that transgenes often do not behave as independent units, but are significantly and variably influenced by a number of factors leading to marked variations in expression patterns between different transgenic lines carrying the same construct [38]. For example, differences in mRNA and protein expression may occur due to the impact of enhancers that regulate neighbouring genes located in close proximity to the inserted transgene [39]. Although these enhancers normally regulate their respective associated gene, they can also affect the expression pattern of a transgene that is inserted near them. In addition, microinjection has the potential to insert multiple copies of a transgene into the recipient genome [28,40], which could lead to distinct copy number profiles within the two independently generated transgenic lines. Previous studies in other transgenic models have shown that copy number influences transgene expression resulting in differential phenotypic effects [41,42]. Another potential factor that could impact on the level of transgene expression is age-related increases in DNA-methylation [43]. For example, previous studies in rodent and pig transgenic models have revealed consistent age-related increases in DNA-methylation of ribosomal genes that correlated with inhibition of gene expression [41,44]. Future investigations will be necessary to determine if any of these factors play a role in the unique expression profiles between SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats.

Both transgenic lines demonstrated an increase in total and transgene specific Gstm1 mRNA expression in kidneys at 5 weeks of age. However, this increased expression was not sustained at the later time point investigated (i.e. 21 weeks of age) in the SHRSP-Tg(Gstm1)2WKY line. This return to SHRSP expression levels at 21 weeks is paralleled by renal protein expression in both SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats. An age-related decline in transgene expression is not unique to this investigation since previously published studies have shown that transgene expression levels can decline with time both in vivo and in vitro[45]. For example, enhanced green fluorescent protein (EGFP) expression, under the control of the EF-1α promoter, was found to be progressively limited during the later stages of development [45], and completely restricted in adult tissue of Xenopus leavis[46], medaka fish [47] and zebrafish [43]. Although these studies have reported a decrease in transgene expression levels during later stages of life, in line with the present findings, each of these studies demonstrate significant phenotypic effects as a result of early transgene expression [43,45–47]. Our data suggest that enhanced expression of Gstm1 in the kidney prior to the onset of hypertension prevents the progression of hypertension in the transgenic SHRSP rat.

The kidney is highly vulnerable to the damage caused by reactive oxygen species (ROS), which can impact on its critical role in salt and water homeostasis, leading to altered renal vascular function and the development of hypertension [48,49]. In this study, we have demonstrated that the BP lowering effects of enhanced Gstm1 expression are paralleled by reduced oxidative damage in the kidney. Specifically, Gstm1 transgenic rats show significantly lower renal lipid peroxidation and reduced renal pathology as indicated by the absence of renal vessel hyperplasia and significantly reduced levels of proteinuria when compared with the SHRSP. The oxidative stress pathway is a complex cascade of events involving multiple types of ROS and antioxidant enzymes. We examined several components of this cascade, namely superoxide (O2), hydrogen peroxide (H2O2), GSSG : GSH ratio and lipid peroxidation. Our data show that ROS generated early in the oxidative stress pathway (i.e. before point at which glutathione acts in the ROS cascade), are not significantly different in kidneys from SHRSP, WKY and transgenic rats. However, later components of the pathway (GSSG : GSH ratio and lipid peroxidation) are reduced in kidneys from the transgenic rats when compared with SHRSP. In contrast, previous studies in 20-week-old SHRSP rats demonstrated significantly increased basal and NADH stimulated O2 levels in renal cortex when compared with WKY rats [50]. Several factors may contribute to this difference in O2 levels between the two studies. For example, there are differences in the methods of O2 measurement between the studies, and whole kidney were used in the current study as opposed to renal cortex. However, similar to the current findings, there were no significant differences observed in renal H2O2 levels between SHRSP and WKY rats at 20 weeks of age. In addition to the 21-week time point we also examined ROS levels in kidneys from rats at 5 weeks of age. Our data showed that there was no evidence of oxidative stress at this early time point in all four strains when compared with kidneys from 21-week-old rats. Therefore, improved Gstm1 expression levels are evident in SHRSP-Tg(Gstm1)1WKY and SHRSP-Tg(Gstm1)2WKY rats before obvious differences in ROS levels or the onset of hypertension.

In parallel to the effect on BP, echocardiography measurements at 21 weeks of age demonstrate that cardiac hypertrophy (i.e. LVMI, relative wall thickness) and cardiac function parameters (i.e. SV, CO) were significantly improved in both transgenic lines compared with the SHRSP. In line with these cardiac changes, Gstm1 mRNA expression levels were significantly increased in WKY hearts compared with SHRSP, with increased trends in expression in the transgenic rats. Currently it is not possible to determine whether the cardiac mass and function changes are due to direct effects of altered Gstm1 expression in the heart or are a secondary effect of the significantly lowered BP in the transgenic animals and further investigation will be required to dissect these factors.

In conclusion, the production of two independently generated Gstm1 transgenic lines on the SHRSP genetic background has provided a unique opportunity to investigate the impact of Gstm1 deficiency on the development of hypertension and oxidative stress. This data supports the hypothesis that reduced renal Gstm1 plays an important role in oxidative stress mechanisms underlying the development of hypertension and end organ damage. The clinical impact of the loss of GSTM1 has recently been demonstrated in a subset of participants from the Atherosclerosis Risk in Communities Study [51]. In this community-based prospective cohort of black and white patients, zero or one copy of GSTM1 was significantly and independently associated with higher risk of kidney failure and heart failure. These results suggest that GSMT1 is a potential therapeutic target that warrants further detailed investigation, and our novel Gstm1 transgenic SHRSP lines will provide an important experimental model for these future preclinical studies.

Back to Top | Article Outline

ACKNOWLEDGEMENTS

The current work was supported by an EU community's Seventh Framework Programme (FP7/2007-2013) under grant agreement (Health-F4-2010-241504 EURATRANS) awarded to A.F.D. A 4-year British Heart Foundation PhD studentship awarded to D.G. (FS/09/052/28032), and the British Heart Foundation Centre of Excellence award (RE/13/5/30177). M.P. was supported by a Praemium Academiae award of the Czech Academy of Sciences and grant 14-36804G from the Czech Science Foundation.

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

REFERENCES

1. Lifton RP, Jeunemaitre X. Finding genes that cause human hypertension. J Hypertens 1993; 11:231–236.
2. Vattikuti S, Guo J, Chow CC. Heritability and genetic correlations explained by common SNPs for metabolic syndrome traits. PLoS Genet 2012; 8:e1002637.
3. He J, Kelly TN, Zhao Q, Li H, Huang J, Wang L, et al. Genome-wide association study identifies 8 novel loci associated with blood pressure responses to interventions in Han Chinese. Circ Cardiovasc Genet 2013; 6:598–607.
4. Cabrera CP, Ng FL, Warren HR, Barnes MR, Munroe PB, Caulfield MJ. Exploring hypertension genome-wide association studies findings and impact on pathophysiology, pathways, and pharmacogenetics. Wiley Interdiscip Rev Syst Biol Med 2015; 7:73–90.
5. Levy D, Ehret GB, Rice K, Verwoert GC, Launer LJ, Dehghan A, et al. Genome-wide association study of blood pressure and hypertension. Nat Genet 2009; 41:677–687.
6. Padmanabhan S, Newton-Cheh C, Dominiczak AF. Genetic basis of blood pressure and hypertension. Trends Genet 2012; 28:397–408.
7. Franceschini N, Reiner AP, Heiss G. Recent findings in the genetics of blood pressure and hypertension traits. Am J Hypertens 2011; 24:392–400.
8. Ganesh SK, Tragante V, Guo W, Guo Y, Lanktree MB, Smith EN, et al. Loci influencing blood pressure identified using a cardiovascular gene-centric array. Hum Mol Genet 2013; 22:1663–1678.
9. Ehret GB, Munroe PB, Rice KM, Bochud M, Johnson AD, Chasman DI, et al. Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. Nature 2011; 478:103–109.
10. Newton-Cheh C, Johnson T, Gateva V, Tobin MD, Bochud M, Coin L, et al. Genome-wide association study identifies eight loci associated with blood pressure. Nat Genet 2009; 41:666–676.
11. Johnson T, Gaunt TR, Newhouse SJ, Padmanabhan S, Tomaszewski M, Kumari M, et al. Blood pressure loci identified with a gene-centric array. Am J Hum Genet 2011; 89:688–700.
12. Zhu X, Young JH, Fox E, Keating BJ, Franceschini N, Kang S, et al. Combined admixture mapping and association analysis identifies a novel blood pressure genetic locus on 5p13: contributions from the CARe consortium. Hum Mol Genet 2011; 20:2285–2295.
13. Tragante V, Barnes MR, Ganesh SK, Lanktree MB, Guo W, Franceschini N, et al. Gene-centric meta-analysis in 87,736 individuals of European ancestry identifies multiple blood-pressure-related loci. Am J Hum Genet 2014; 94:349–360.
14. Warren HR, Evangelou E, Cabrera CP, Gao H, Ren M, Mifsud B, et al. Genome-wide association analysis identifies novel blood pressure loci and offers biological insights into cardiovascular risk. Nat Genet 2017; 49:403–415.
15. Ehret GB, Ferreira T, Chasman DI, Jackson AU, Schmidt EM, Johnson T, et al. The genetics of blood pressure regulation and its target organs from association studies in 342,415 individuals. Nat Genet 2016; 48:1171–1184.
16. Lerman LO, Chade AR, Sica V, Napoli C. Animal models of hypertension: an overview. J Lab Clin Med 2005; 146:160–173.
17. Delles C, McBride MW, Graham D, Padmanabhan S, Dominiczak AF. Genetics of hypertension: from experimental animals to humans. Biochim Biophys Acta 2010; 1802:1299–1308.
18. Yamori Y, Horie R, Handa H, Sato M, Fukase M. Pathogenetic similarity of strokes in stroke-prone spontaneously hypertensive rats and humans. Stroke 1976; 7:46–53.
19. Conrad CH, Brooks WW, Robinson KG, Bing OH. Impaired myocardial function in spontaneously hypertensive rats with heart failure. Am J Physiol 1991; 260 (1 Pt 2):H136–H145.
20. Clark JS, Jeffs B, Davidson AO, Lee WK, Anderson NH, Bihoreau MT, et al. Quantitative trait loci in genetically hypertensive rats. Possible sex specificity. Hypertension 1996; 28:898–906.
21. Stranger BE, Stahl EA, Raj T. Progress and promise of genome-wide association studies for human complex trait genetics. Genetics 2011; 187:367–383.
22. Koh-Tan HH, McBride MW, McClure JD, Beattie E, Young B, Dominiczak AF, et al. Interaction between chromosome 2 and 3 regulates pulse pressure in the stroke-prone spontaneously hypertensive rat. Hypertension 2013; 62:33–40.
23. Jeffs B, Negrin CD, Graham D, Clark JS, Anderson NH, Gauguier D, Dominiczak AF. Applicability of a ‘speed’ congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2. Hypertension 2000; 35 (1 Pt 2):179–187.
24. McBride MW, Carr FJ, Graham D, Anderson NH, Clark JS, Lee WK, et al. Microarray analysis of rat chromosome 2 congenic strains. Hypertension 2003; 41 (3 Pt 2):847–853.
25. McBride MW, Brosnan MJ, Mathers J, McLellan LI, Miller WH, Graham D, et al. Reduction of Gstm1 expression in the stroke-prone spontaneously hypertension rat contributes to increased oxidative stress. Hypertension 2005; 45:786–792.
26. Nebert DW, Vasiliou V. Analysis of the glutathione S-transferase (GST) gene family. Hum Genomics 2004; 1:460–464.
27. Pontén F, Jirström K, Uhlen M. The human protein atlas – a tool for pathology. J Pathol 2008; 216:387–393.
28. Pravenec M, Landa V, Zidek V, Musilova A, Kren V, Kazdova L, et al. Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats. Nat Genet 2001; 27:156–158.
29. Davidson AO, Schork N, Jaques BC, Kelman AW, Sutcliffe RG, Reid JL, Dominiczak AF. Blood pressure in genetically hypertensive rats. Influence of the Y chromosome. Hypertension 1995; 26:452–459.
30. Graham D, Hamilton C, Beattie E, Spiers A, Dominiczak AF. Comparison of the effects of omapatrilat and irbesartan/hydrochlorothiazide on endothelial function and cardiac hypertrophy in the stroke-prone spontaneously hypertensive rat: sex differences. J Hypertens 2004; 22:329–337.
31. Masson R, Nicklin SA, Craig MA, McBride M, Gilday K, Gregorevic P, et al. Onset of experimental severe cardiac fibrosis is mediated by overexpression of angiotensin-converting enzyme 2. Hypertension 2009; 53:694–700.
32. Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun 2018; 9:1911.
33. Vu DM, Shaughnessy J, Lewis LA, Ram S, Rice PA, Granoff DM. Enhanced bacteremia in human factor H transgenic rats infected by Neisseria meningitidis. Infect Immun 2012; 80:643–650.
34. Landa V, Zídek V, Mlejnek P, Šimáková M, Šilhavý J, Trnovská J, et al. Sterol regulatory element binding protein 2 overexpression is associated with reduced adipogenesis and ectopic fat accumulation in transgenic spontaneously hypertensive rats. Physiol Res 2014; 63:587–590.
35. Hadar R, Edemann-Callesen H, Reinel C, Wieske F, Voget M, Popova E, et al. Rats overexpressing the dopamine transporter display behavioral and neurobiological abnormalities with relevance to repetitive disorders. Sci Rep 2016; 6:39145.
36. Škop V, Trnovská J, Oliyarnyk O, Marková I, Malínská H, Kazdová L, et al. Hepatotoxic effects of fenofibrate in spontaneously hypertensive rats expressing human C-reactive protein. Physiol Res 2016; 65:891–899.
37. Manakov D, Kolar D, Zurmanova J, Pravenec M, Novotny J. Changes in the activity of some metabolic enzymes in the heart of SHR rat incurred by transgenic expression of CD36. J Physiol Biochem 2018; 74:479–489.
38. Robertson G, Garrick D, Wilson M, Martin DI, Whitelaw E. Age-dependent silencing of globin transgenes in the mouse. Nucleic Acids Res 1996; 24:1465–1471.
39. Clark AJ, Bissinger P, Bullock DW, Damak S, Wallace R, Whitelaw CB, Yull F. Chromosomal position effects and the modulation of transgene expression. Reprod Fertil Dev 1994; 6:589–598.
40. Pravenec M, Landa V, Zídek V, Musilová A, Kazdová L, Qi N, et al. Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension. Physiol Res 2003; 52:681–688.
41. Kong Q, Wu M, Huan Y, Zhang L, Liu H, Bou G, et al. Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs. PLoS One 2009; 4:e6679.
42. Pérez VI, Cortez LA, Lew CM, Rodriguez M, Webb CR, Van Remmen H, et al. Thioredoxin 1 overexpression extends mainly the earlier part of life span in mice. J Gerontol A Biol Sci Med Sci 2011; 66:1286–1299.
43. Thummel R, Burket CT, Hyde DR. Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration. ScientificWorldJournal 2006; 6 (Suppl 1):65–81.
44. Calero-Nieto FJ, Bert AG, Cockerill PN. Transcription-dependent silencing of inducible convergent transgenes in transgenic mice. Epigenetics Chromatin 2010; 3:3.
45. Ueda Y, Mizuno N, Araki M. Transgenic Xenopus laevis with the ef1-α promoter as an experimental tool for amphibian retinal regeneration study. Genesis 2012; 50:642–650.
46. Gross JB, Hanken J, Oglesby E, Marsh-Armstrong N. Use of a ROSA26:GFP transgenic line for long-term Xenopus fate-mapping studies. J Anat 2006; 209:401–413.
47. Kinoshita M, Kani S, Ozato K, Wakamatsu Y. Activity of the medaka translation elongation factor 1alpha-A promoter examined using the GFP gene as a reporter. Dev Growth Differ 2000; 42:469–478.
48. Ozbek E. Induction of oxidative stress in kidney. Int J Nephrol 2012; 2012:465897.
49. Shimosawa T, Mu S, Shibata S, Fujita T. The kidney and hypertension: pathogenesis of salt-sensitive hypertension. Curr Hypertens Rep 2012; 14:468–472.
50. Koh-Tan HH, Graham D, Hamilton CA, Nicoll G, Fields L, McBride MW, et al. Renal and vascular glutathione S-transferase mu is not affected by pharmacological intervention to reduce systolic blood pressure. J Hypertens 2009; 27:1575–1584.
51. Tin A, Scharpf R, Estrella MM, Yu B, Grove ML, Chang PP, et al. The loss of GSTM1 associates with kidney failure and heart failure. J Am Soc Nephrol 2017; 28:3345–3352.

* Martin W. McBride and Delyth Graham contributed equally to the article.

Keywords:

glutathione S-transferase μ-type 1; hypertension; oxidative stress; stroke-prone spontaneously hypertensive rat; transgenic

Supplemental Digital Content

Back to Top | Article Outline
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.