Essential or primary hypertension is a complex polygenic disease with geneticheritability averaging approximately 30% and with strong influence ofenvironmental factors and gene-environment interaction. Heterogeneity in thegeneral population and the polygenic complexities of the disease has meant thatidentification and functional validation of candidate genes has proved extremelydifficult in humans. Several strategies have been developed to dissect geneticdeterminants of hypertension, one of which is the use of rodent models (1;2).Animal models of heritable hypertension offer more favourable investigativeopportunities because of reduced genetic heterogeneity, the capacity forcontrolled breeding and environmental conditions, and the ability to producegenetic crosses and analyse large numbers of progeny. The stroke-pronespontaneously hypertensive rat (SHRSP) is a commonly used model of humanessential hypertension. Previous studies conducted in our laboratory utilizing acombination of congenic strain construction and genome-wide microarrayexpression profiling in the SHRSP have allowed us to identify the positionalcandidate gene, glutathione S-transferase μ-type 1 (Gstm1), which is involved inthe defence against oxidative stress and is significantly down-regulated in theSHRSP (3;4). Genomic DNA sequencing of Gstm1 in SHRSP and WKY identified 13single nucleotide polymorphisms (SNPs), an insertion and a deletion (5).Luciferase reporter gene assays implicated five SNPs to be responsible forsignificant reduction in luciferase activity measurements (6). In consideration ofthese previous studies, it is hypothesized that Gstm1 deficiency in the SHRSPplays a causative role in the development of oxidative stress and hypertension.To establish definitive proof that reduced Gstm1 expression affects bloodpressure regulation and oxidative stress, two independent transgenic lines(referred to as Trans1 and Trans2) of SHRSP were created with the aim ofrescuing Gstm1 deficiency by incorporation of a normal Gstm1 gene into theSHRSP genome. Generation of these transgenic SHRSP rats involvedmicroinjection with a 2.7 kb linear construct encoding wild type (WKY) Gstm1under the control of the universal EF-1α promoter. They were generated usingthe same expression platform and microinjection fragment purification protocolemployed in the successful production of the CD-36 transgenic, rat as previouslydescribed (7). The transgenic protocol was carried out in collaboration with Dr17Michal Pravenec (Prague), who is an expert in transgenic rat production, usingmale and female SHRSP rats from the University of Glasgow colony.Oxidative stress is an important pathogenic factor in the development ofcardiovascular disease. Glutathione S-transferases protect against oxidativestress-induced injury through the detoxification of reactive oxygen species. It ishypothesised that Gstm1 deficiency in the SHRSP plays a causative role in thedevelopment of oxidative stress and hypertension. Thus the aims of this studywere to establish definitive proof that reduced Gstm1 expression in the SHRSPplays a causative role in the development of hypertension and oxidative stressthrough utilizing a combinational approach of in vivo and ex vivo studiesalongside molecular analysis to fully characterize the Gstm1 transgenic SHRSPrat. Additionally, information and insights gained from this investigation from theGstm1 transgenic SHRSP will be applied to a translation aspect for theinvestigation of GSTM family in humans.Functional validation through hemodynamic and cardiac analysis includedmeasurement of systolic, diastolic and mean arterial blood pressures, pulsepressure and heart rate using the Dataquest IV telemetry system (Data SciencesInternational) and transthoracic echocardiography was used to assess cardiacgeometry and contractility. Telemetry data show that there is a significantreduction in systolic blood pressure, diastolic blood pressures, and pulse pressurein both of the transgenic lines when compared to the SHRSP suggesting thatincorporation of a WKY type Gstm1 gene into the SHRSP genome does indeedreduce the hypertensive phenotype. Moreover, the observed reduction in systolicblood pressure is remarkably similar in magnitude to that demonstrated in theChromosome 2 congenic strain, SP.WKYGla2c*, in which Gstm1 was identified asa candidate gene for hypertension. In order to investigate the potential role ofGstm1 deficiency in the salt-sensitivity phenotype in SHRSP rats, parental strainrats 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 theSHRSP, however, the Trans1 line still maintained a significant decrease in systolicand diastolic blood pressure compared to the SHRSP during salt loading.18In parallel with the significantly lower SBP, DBP and PP we also observesignificantly improved cardiac function and reduced cardiac hypertrophy in thetwo independently generated transgenic lines. While there was no significantchanges in both fractional shortening (FS) and ejection fraction (EF), betweenthe four strains, relative wall thickness was significantly reduced in WKY, Trans1,and Trans2 rats when compared to the SHRSP with Trans1 and Trans2 ratsshowing an intermediate phenotype between the parental strains.Analysis of genetic and molecular changes resulting from the random insertion ofGstm1 into the SHRSP genome included assessment of transgene (WKY form) andtotal Gstm1 gene expression, protein quantification, immunohistochemistry(IHC), transgene insertion and copy number. Both transgenic lines demonstratedan increase in total and transgene specific expression of Gstm1 in kidneys at 5weeks of age as well as increased transgene expression in several othercardiovascular tissues. Protein expression was also similarly increased in thekidney at 5 weeks of age and showed a similar expression pattern to that of theWKY. Additionally, we saw increased total Gstm1 expression in a range ofcardiovascular tissues at 21 weeks of age without changes of other Gstm familymembers (Gstm2 and Gstm3). Although it was not possible to identify the exactlocation of the transgene insertion site in both transgenic lines, data presentedindicate that they are not identically inserted. Furthermore, sequencing datashows that each transgenic line contains multiple copies of the transgene acrossa number of generations.To assess renal function in the Gstm1 transgenic lines, rats from each line thatwere implanted with telemetry probes were assessed by 24-hr metabolic cagemeasurements which allowed for analysis of indirect glomerular filtration ratealong with proteinuria and urinary electrolyte measurements. Histologicalanalysis was used to assess renal morphology by examining haematoxylin andeosin (H&E) stained sections. Fibrosis was examined by staining with picrosiriusred. At 21 weeks, we saw evidence of reduced renal pathology as indicated bythe absence of renal vessel hyperplasia and reduced proteinuria in the WKY,Trans1, and Trans2 rats. H&E staining showed a more similar morphology to theWKY in the transgenic lines with no signs of accelerated hypertension. Theseimprovements in renal pathology were also apparent in salt-loaded Trans1 rats.19Oxidative stress and myography measurements were also carried out in order toascertain the impact of increased Gstm1 expression on the SHRSP geneticbackground. The data presented in this study clearly shows a reduction in renaloxidative stress in both transgenic lines. Furthermore, these improvements inoxidative stress were also apparent in salt-loaded Trans1 rats. Aortic andmesenteric artery wire myography data showed that there was no significantdifference between SHRSP and transgenic lines for vascular function. Pressuremyography in the mesenteric arteries, demonstrated that the transgenic lineswere only significantly different from the SHRSP in terms of increased vesselcross sectional area (CSA), but did show trends of improved structure andmechanical alterations of these vessels.Additionally, we investigated the rodent GSTM family and applied them to ahuman cohort in order to assess translational aspects and expanded the studypreviously conducted by Delles et al. (8). The investigation included a largernumber of subjects than that studied previously by Delles et al. and allowed forthe elucidation of the relationship between other members of the GSTM familyand human essential hypertension. While there was no significant difference inrenal expression in GSTM5, GSTM3, GPx-1 and GPx-3 between normotensive andhypertensive patient, we found that GSTM2 expression was significantlyincreased in hypertensive patients when compared to normotensive patients.Moreover, we found there to be a borderline significance (p=0.054) between thers11802 SNP genotype and GSTM5 expression. Further investigation between geneexpression correlations showed there was a significant linear correlationbetween GSTM5 and GPx-1.In summary, multiple phenotypic and molecular techniques were applied in theanalysis of the GSTM1 transgenic SHRSP. Transgenic SHRSP rats expressing theWKY form of the GSTM1 gene demonstrate significantly reduced blood pressure,oxidative stress and improved levels of renal GSTM1 expression. This datasupports the hypothesis that significantly reduced renal GSTM1 plays a causativerole in the development of hypertension in the SHRSP rat.
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Characterization of the GSTM1 transgenic SHRSP rat
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