Characterization of the GSTM1 transgenic SHRSP rat

Olson, Erin D. (2014) Characterization of the GSTM1 transgenic SHRSP rat. PhD thesis, University of Glasgow.

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Abstract

Essential or primary hypertension is a complex polygenic disease with genetic
heritability averaging approximately 30% and with strong influence of
environmental factors and gene-environment interaction. Heterogeneity in the
general population and the polygenic complexities of the disease has meant that
identification and functional validation of candidate genes has proved extremely
difficult in humans. Several strategies have been developed to dissect genetic
determinants of hypertension, one of which is the use of rodent models (1;2).
Animal models of heritable hypertension 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. The stroke-prone
spontaneously hypertensive rat (SHRSP) is a commonly used model of human
essential hypertension. Previous studies conducted in our laboratory utilizing a
combination of congenic strain construction and genome-wide microarray
expression profiling in the SHRSP have allowed us to identify the positional
candidate gene, glutathione S-transferase μ-type 1 (Gstm1), which is involved in
the defence against oxidative stress and is significantly down-regulated in the
SHRSP (3;4). Genomic DNA sequencing of Gstm1 in SHRSP and WKY identified 13
single nucleotide polymorphisms (SNPs), an insertion and a deletion (5).
Luciferase reporter gene assays implicated five SNPs to be responsible for
significant reduction in luciferase activity measurements (6). In consideration of
these previous studies, it is hypothesized that Gstm1 deficiency in the SHRSP
plays a causative role in the development of oxidative stress and hypertension.
To establish definitive proof that reduced Gstm1 expression affects blood
pressure regulation and oxidative stress, two independent transgenic lines
(referred to as Trans1 and Trans2) of SHRSP were created with the aim of
rescuing Gstm1 deficiency by incorporation of a normal Gstm1 gene into the
SHRSP genome. Generation of these transgenic SHRSP rats involved
microinjection with a 2.7 kb linear construct encoding wild type (WKY) Gstm1
under the control of the universal EF-1α promoter. They were generated using
the same expression platform and microinjection fragment purification protocol
employed in the successful production of the CD-36 transgenic, rat as previously
described (7). The transgenic protocol was carried out in collaboration with Dr
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Michal Pravenec (Prague), who is an expert in transgenic rat production, using
male and female SHRSP rats from the University of Glasgow colony.
Oxidative stress is an important pathogenic factor in the development of
cardiovascular disease. Glutathione S-transferases protect against oxidative
stress-induced injury through the detoxification of reactive oxygen species. It is
hypothesised that Gstm1 deficiency in the SHRSP plays a causative role in the
development of oxidative stress and hypertension. Thus the aims of this study
were to establish definitive proof that reduced Gstm1 expression in the SHRSP
plays a causative role in the development of hypertension and oxidative stress
through utilizing a combinational approach of in vivo and ex vivo studies
alongside molecular analysis to fully characterize the Gstm1 transgenic SHRSP
rat. Additionally, information and insights gained from this investigation from the
Gstm1 transgenic SHRSP will be applied to a translation aspect for the
investigation of GSTM family in humans.
Functional validation through hemodynamic and cardiac analysis included
measurement of systolic, diastolic and mean arterial blood pressures, pulse
pressure and heart rate using the Dataquest IV telemetry system (Data Sciences
International) and transthoracic echocardiography was used to assess cardiac
geometry and contractility. Telemetry data show that there is a significant
reduction in systolic blood pressure, diastolic blood pressures, and pulse pressure
in both of the transgenic lines when compared to the SHRSP suggesting that
incorporation of a WKY type Gstm1 gene into the SHRSP genome does indeed
reduce the hypertensive phenotype. Moreover, the observed reduction in systolic
blood pressure is remarkably similar in magnitude to that demonstrated in the
Chromosome 2 congenic strain, SP.WKYGla2c*, in which Gstm1 was identified as
a candidate gene for hypertension. In order to investigate the potential role of
Gstm1 deficiency in the salt-sensitivity phenotype in SHRSP rats, parental strain
rats and Trans1 animals underwent 1% salt loading starting at 18 weeks of age.
This resulted in Trans1 displaying a trend towards salt-sensitivity (i.e.
exaggerated night-time daytime blood pressure variation) similar to that of the
SHRSP, however, the Trans1 line still maintained a significant decrease in systolic
and diastolic blood pressure compared to the SHRSP during salt loading.
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In parallel with the significantly lower SBP, DBP and PP we also observe
significantly improved cardiac function and reduced cardiac hypertrophy in the
two independently generated transgenic lines. While there was no significant
changes in both fractional shortening (FS) and ejection fraction (EF), between
the four strains, relative wall thickness was significantly reduced in WKY, Trans1,
and Trans2 rats when compared to the SHRSP with Trans1 and Trans2 rats
showing an intermediate phenotype between the parental strains.
Analysis of genetic and molecular changes resulting from the random insertion of
Gstm1 into the SHRSP genome included assessment of transgene (WKY form) and
total Gstm1 gene expression, protein quantification, immunohistochemistry
(IHC), transgene insertion and copy number. Both transgenic lines demonstrated
an increase in total and transgene specific expression of Gstm1 in kidneys at 5
weeks of age as well as increased transgene expression in several other
cardiovascular tissues. Protein expression was also similarly increased in the
kidney at 5 weeks of age and showed a similar expression pattern to that of the
WKY. Additionally, we saw increased total Gstm1 expression in a range of
cardiovascular tissues at 21 weeks of age without changes of other Gstm family
members (Gstm2 and Gstm3). Although it was not possible to identify the exact
location of the transgene insertion site in both transgenic lines, data presented
indicate that they are not identically inserted. Furthermore, sequencing data
shows that each transgenic line contains multiple copies of the transgene across
a number of generations.
To assess renal function in the Gstm1 transgenic lines, rats from each line that
were implanted with telemetry probes were assessed by 24-hr metabolic cage
measurements which allowed for analysis of indirect glomerular filtration rate
along with proteinuria and urinary electrolyte measurements. Histological
analysis was used to assess renal morphology by examining haematoxylin and
eosin (H&E) stained sections. Fibrosis was examined by staining with picrosirius
red. At 21 weeks, we saw evidence of reduced renal pathology as indicated by
the absence of renal vessel hyperplasia and reduced proteinuria in the WKY,
Trans1, and Trans2 rats. H&E staining showed a more similar morphology to the
WKY in the transgenic lines with no signs of accelerated hypertension. These
improvements in renal pathology were also apparent in salt-loaded Trans1 rats.
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Oxidative stress and myography measurements were also carried out in order to
ascertain the impact of increased Gstm1 expression on the SHRSP genetic
background. The data presented in this study clearly shows a reduction in renal
oxidative stress in both transgenic lines. Furthermore, these improvements in
oxidative stress were also apparent in salt-loaded Trans1 rats. Aortic and
mesenteric artery wire myography data showed that there was no significant
difference between SHRSP and transgenic lines for vascular function. Pressure
myography in the mesenteric arteries, demonstrated that the transgenic lines
were only significantly different from the SHRSP in terms of increased vessel
cross sectional area (CSA), but did show trends of improved structure and
mechanical alterations of these vessels.
Additionally, we investigated the rodent GSTM family and applied them to a
human cohort in order to assess translational aspects and expanded the study
previously conducted by Delles et al. (8). The investigation included a larger
number of subjects than that studied previously by Delles et al. and allowed for
the elucidation of the relationship between other members of the GSTM family
and human essential hypertension. While there was no significant difference in
renal expression in GSTM5, GSTM3, GPx-1 and GPx-3 between normotensive and
hypertensive patient, we found that GSTM2 expression was significantly
increased in hypertensive patients when compared to normotensive patients.
Moreover, we found there to be a borderline significance (p=0.054) between the
rs11802 SNP genotype and GSTM5 expression. Further investigation between gene
expression correlations showed there was a significant linear correlation
between GSTM5 and GPx-1.
In summary, multiple phenotypic and molecular techniques were applied in the
analysis of the GSTM1 transgenic SHRSP. Transgenic SHRSP rats expressing the
WKY form of the GSTM1 gene demonstrate significantly reduced blood pressure,
oxidative stress and improved levels of renal GSTM1 expression. This data
supports the hypothesis that significantly reduced renal GSTM1 plays a causative
role in the development of hypertension in the SHRSP rat.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Kidney, Gstm1, Hypertension
Subjects: Q Science > QH Natural history > QH426 Genetics
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences > Cardiovascular Science
Supervisor's Name: Delyth , Dr. Graham
Date of Award: June 2014
Depositing User: Mrs. Erin Olson
Unique ID: glathesis:2014-5272
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 31 Jul 2014 11:52
Last Modified: 31 Jul 2014 11:52
URI: http://theses.gla.ac.uk/id/eprint/5272

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