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 17 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. 18 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. 19 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:616428 |
| Date | January 2014 |
| Creators | Olson, Erin D. |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5272/ |
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