Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension.

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.

Genetic susceptibility to renal injury in hypertension.

Substantial evidence indicates that hypertension plays a predominant role in the progression of most chronic renal diseases including diabetic nephropathy. Nevertheless, significant differences are observed in the susceptibility to develop hypertension-associated renal damage between individuals, racial groups and animal strains despite comparable hypertension. Recent studies employing a variety of genetic methods both in humans and in experimental models, have provided strong support for the potential importance of genetic factors and have suggested that genes influencing susceptibility to renal damage may be inherited separately from genes that influence blood pressure. However, due to the genetic complexity involved in a multifactorial trait such as the susceptibility to hypertensive renal damage, very limited progress has been achieved thus far in attempts to link such susceptibility to specific genetic mechanisms, chromosome regions and/or candidate genes. It is anticipated that the rapid recent advances in molecular genetic techniques and the simultaneous use of multiple complementary strategies, as is currently under way, will greatly facilitate this search and provide fundamental new insights into the pathogenesis of hypertensive renal damage.

Genetic isolation of a blood pressure quantitative trait locus on chromosome 2 in the spontaneously hypertensive rat.

OBJECTIVES: Total genome scans of genetically segregating populations derived from the spontaneously hypertensive rat (SHR) and other rat models of hypertension have suggested the presence of quantitative trait loci (QTL) regulating blood pressure and cardiac mass on multiple chromosomes, including chromosome 2. The objective of the current study was to directly test for the presence of a blood pressure QTL on rat chromosome 2. DESIGN: A new congenic strain was derived by replacing a segment of chromosome 2 in the SHR between D2Rat171 and D2Arb24 with the corresponding chromosome segment from the normotensive Brown Norway rat. Arterial pressures were directly monitored in conscious rats by radiotelemetry. RESULTS: We found that the SHR congenic strain (SHR-2) carrying a segment of chromosome 2 from the Brown Norway rat had significantly lower systolic and diastolic blood pressures than the SHR progenitor strain. The attenuation of hypertension in the SHR-2 congenic strain versus the SHR progenitor strain was accompanied by significant amelioration of cardiac hypertrophy. CONCLUSIONS: These findings demonstrate that gene(s) with major effects on blood pressure exist in the differential segment of chromosome 2 trapped within the new SHR.BN congenic strain.

Y-chromosome transfer induces changes in blood pressure and blood lipids in SHR.

Previous studies with chromosome-Y consomic strains of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats suggest that a quantitative trait locus for blood pressure regulation exists on chromosome Y. To test this hypothesis in the SHR-Brown Norway (BN) model and to study the effects of chromosome Y on lipid and carbohydrate metabolism, we produced a new consomic strain of SHR carrying the Y chromosome transferred from the BN rat. We found that replacing the SHR Y chromosome with the BN Y chromosome resulted in significant decreases in systolic and diastolic blood pressures in the SHR.BN-Y consomic strain (P<0.05). To elicit possible dietary-induced variation in lipid and glucose metabolism between the SHR progenitor and chromosome-Y consomic strains, we fed rats a high-fructose diet for 15 days in addition to the normal diet. On the high-fructose diet, the SHR.BN-Y consomic rats exhibited significantly increased levels of serum triglycerides and decreased levels of serum HDL cholesterol versus the SHR progenitor rats. Glucose tolerance and insulin/glucose ratios, however, were similar in both strains on both normal and high-fructose diets. These findings provide direct evidence that a gene or genes on chromosome Y contribute to the pathogenesis of spontaneous hypertension in the SHR-BN model. These results also indicate that transfer of the Y chromosome from the BN rat onto the SHR background exacerbates dietary-induced dyslipidemia in SHR. Thus, genetic variation in genes on the Y chromosome may contribute to variation in blood pressure and lipid levels and may influence the risk for cardiovascular disease.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Hypoalgesia and hyperalgesia with inherited hypertension in the rat.

Many studies indicate that blood pressure control systems can attenuate pain (hypoalgesia) of short duration; however, we recently found exaggerated nociceptive responses (hyperalgesia) of persistent duration in the spontaneously hypertensive rat (SHR). Here, we used SHR, Dahl Salt-Sensitive (SS), and normotensive control rats to evaluate the contribution of sustained elevations in arterial pressure to nociceptive responses. Compared with Sprague-Dawley and/or Wistar-Kyoto controls, SHR were 1) hypoalgesic in the hot plate test and 2) hyperalgesic in longer latency tail and paw-withdrawal tests and in two models of inflammatory nociception. These differences were not observed between SS and salt-resistant controls fed a high-salt diet. Inflammatory hyperalgesia in SHR was correlated with neither paw edema nor the number of Fos-positive spinal cord neurons. Our results indicate that "pain" phenotype of the SHR is not restricted to hypoalgesia. This phenotype is related to genetic factors or to the autonomic systems that control blood pressure and not to sustained elevations in blood pressure, differences in spinal neuron activity, or inflammatory edema.

Genetic analysis of rat chromosome 1 and the Sa gene in spontaneous hypertension.

Linkage studies in segregating populations derived from the spontaneously hypertensive rat (SHR) indicate that a blood pressure quantitative trait locus exists on rat chromosome 1 in the vicinity of the Sa gene. On the basis of these findings and the observation of increased renal expression of the Sa gene in SHR versus normotensive rats, the Sa gene has been proposed as a candidate gene for spontaneous hypertension. In SHR congenic strains, we and others have found that replacement of a segment of SHR chromosome 1 that contains the Sa gene with the corresponding chromosome segment from a normotensive Brown Norway (BN) rat or Wistar-Kyoto rat can reduce blood pressure. To test whether the Sa gene is necessary for the effect of this region of chromosome 1 on blood pressure, we studied a new SHR congenic subline that harbors a smaller segment of BN chromosome 1 that does not include the Sa gene. Transfer of this subregion of chromosome 1 from the BN rat onto the SHR background was associated with significant reductions in blood pressure comparable to those previously observed on transfer of a larger region of chromosome 1 that included the Sa gene. Thus, in the SHR-BN model of hypertension, the results of these mapping studies (1) demonstrate that molecular variation in the Sa gene is not required for the effect of this region of chromosome 1 on blood pressure and (2) should direct attention toward other candidate genes within the differential chromosome segment of the new congenic subline.

Genetic isolation of a chromosome 1 region affecting susceptibility to hypertension-induced renal damage in the spontaneously hypertensive rat.

Linkage studies in the fawn-hooded hypertensive rat have suggested that genes influencing susceptibility to hypertension-associated renal failure may exist on rat chromosome 1q. To investigate this possibility in a widely used model of hypertension, the spontaneously hypertensive rat (SHR), we compared susceptibility to hypertension-induced renal damage between an SHR progenitor strain and an SHR congenic strain that is genetically identical except for a defined region of chromosome 1q. Backcross breeding with selection for the markers D1Mit3 and Igf2 on chromosome 1 was used to create the congenic strain (designated SHR.BN-D1Mit3/Igf2) that carries a 22 cM segment of chromosome 1 transferred from the normotensive Brown Norway rat onto the SHR background. Systolic blood pressure (by radiotelemetry) and urine protein excretion were measured in the SHR progenitor and congenic strains before and after the induction of accelerated hypertension by administration of DOCA-salt. At the same level of DOCA-salt hypertension, the SHR.BN-D1Mit3/Igf2 congenic strain showed significantly greater proteinuria and histologically assessed renal vascular and glomerular injury than the SHR progenitor strain. These findings demonstrate that a gene or genes that influence susceptibility to hypertension-induced renal damage have been trapped in the differential chromosome segment of the SHR.BN-D1Mit3/Igf2 congenic strain. This congenic strain represents an important new model for the fine mapping of gene(s) on chromosome 1 that affect susceptibility to hypertension-induced renal injury in the rat.

Effect of chromosome 19 transfer on blood pressure in the spontaneously hypertensive rat.

Linkage studies in the spontaneously hypertensive rat (SHR) have suggested that a gene or genes regulating blood pressure may exist on rat chromosome 19 in the vicinity of the angiotensinogen gene. To test this hypothesis, we measured blood pressure in SHR progenitor and congenic strains that are genetically identical except for a segment of chromosome 19 containing the angiotensinogen gene transferred from the normotensive Brown Norway (BN) strain. Transfer of this segment of chromosome 19 from the BN strain onto the genetic background of the SHR induced significant decreases in systolic and diastolic blood pressures in the recipient SHR chromosome 19 congenic strain. To test for differences in angiotensinogen gene expression between the congenic and progenitor strains, we measured angiotensinogen mRNA levels in a variety of tissues, including aorta, brain, kidney, and liver. We found no differences between the progenitor and congenic strains in the angiotensinogen coding sequence or in angiotensinogen expression that would account for the blood pressure differences between the strains. In addition, no significant differences in plasma levels of angiotensinogen or plasma renin activity were detected between the 2 strains. Thus, transfer of a segment of chromosome 19 containing angiotensinogen from the BN rat into the SHR induces a decrease in blood pressure without inducing any major changes in plasma angiotensinogen levels or plasma renin activity. These results indicate that the differential chromosome segment trapped in the SHR chromosome 19 congenic strain contains a quantitative trait locus that influences blood pressure in the SHR but that this blood pressure effect is not explained by differences in plasma angiotensinogen levels or angiotensinogen expression.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Effect of renin gene transfer on blood pressure in the spontaneously hypertensive rat.

To investigate whether molecular variation in the renin gene contributes to the greater blood pressure of spontaneously hypertensive rats (SHR) versus normotensive Brown Norway (BN) rats, we measured blood pressure in an SHR progenitor strain and an SHR congenic strain that are genetically identical except at the renin gene and an associated segment of chromosome 13 transferred from the BN strain. Backcross breeding and molecular selection at the renin locus were used to create the SHR congenic strain (designated SHR.BN-Ren) that carries the renin gene transferred from the normotensive BN strain. We found that transfer of the renin gene from the BN strain onto the genetic background of the SHR did not decrease blood pressure in rats fed either a normal or high-salt diet. In fact, the systolic blood pressures of the SHR congenic rats tended to be slightly greater than the systolic blood pressures of the SHR progenitor rats. However, the congenic strain exhibited lower serum high-density lipoprotein cholesterol, and greater levels of total cholesterol, very-low-density lipoprotein, and intermediate-density lipoprotein cholesterol during administration of a high-fat, high-cholesterol diet. These findings demonstrate that (1) under the environmental circumstances of the current study, the greater blood pressure of SHR versus BN rats cannot be explained by strain differences in the renin gene and (2) a quantitative trait locus affecting lipid metabolism exists on chromosome 13 within the transferred chromosome segment. The SHR.BN-Ren congenic strain may provide a useful new animal model for studying the interaction between high blood pressure and dyslipidemia in cardiovascular disease.

Linkage mapping of the interleukin 1beta converting enzyme (Il1bc) and the glutamate receptor subunit KA1 (Grik4) genes to rat chromosome 8.

Genes for interleukin 1beta converting enzyme (Il1bc) and the glutamate receptor subunit KA1 (Grik4) have been mapped to a centromeric region of rat chromosome 8 using linkage analysis of HXB and BXH recombinant inbred strains. The current results demonstrate that rat chromosome 8 is largely homologous to mouse chromosome 9.

Genetic isolation of a chromosome 1 region affecting blood pressure in the spontaneously hypertensive rat.

Recent linkage studies in the spontaneously hypertensive rat (SHR) suggest that a blood pressure regulatory gene or genes may be located on rat chromosome 1q. To investigate this possibility, we replaced a region of chromosome 1 in the SHR (defined by the markers D1Mit3 and Igf2) with the corresponding chromosome segment from the normotensive Brown-Norway (BN) strain. In male SHR congenic rats carrying the transferred BN chromosome segment, 24-hour average systolic and diastolic blood pressures were significantly lower than in male progenitor SHR. Polymerase chain reaction genotyping using 60 polymorphic microsatellite markers dispersed throughout the genome confirmed the congenic status of the new strain designated SHR.BN-D1Mit3/Igf2. These findings provide direct evidence that a blood pressure regulatory gene exists on the differential segment of chromosome 1 that is sufficient to decrease blood pressure in the SHR. The SHR.BN-D1Mit3/Igf2 congenic strain represents an important new model for fine mapping and characterization of genes on chromosome 1 involved in the pathogenesis of spontaneous hypertension.

Genetic susceptibility to hypertension-induced renal damage in the rat. Evidence based on kidney-specific genome transfer.

To test the hypothesis that genetic factors can determine susceptibility to hypertension-induced renal damage, we derived an experimental animal model in which two genetically different yet histocompatible kidneys are chronically and simultaneously exposed to the same blood pressure profile and metabolic environment within the same host. Kidneys from normotensive Brown Norway rats were transplanted into unilaterally nephrectomized spontaneously hypertensive rats (SHR-RT1.N strain) that harbor the major histocompatibility complex of the Brown Norway strain. 25 d after the induction of severe hypertension with deoxycorticosterone acetate and salt, proteinuria, impaired glomerular filtration rate, and extensive vascular and glomerular injury were observed in the Brown Norway donor kidneys, but not in the SHR-RT1.N kidneys. Control experiments demonstrated that the strain differences in kidney damage could not be attributed to effects of transplantation-induced renal injury, immunologic rejection phenomena, or preexisting strain differences in blood pressure. These studies (a) demonstrate that the kidney of the normotensive Brown Norway rat is inherently much more susceptible to hypertension-induced damage than is the kidney of the spontaneously hypertensive rat, and (b) establish the feasibility of using organ-specific genome transplants to map genes expressed in the kidney that determine susceptibility to hypertension-induced renal injury in the rat.

Mapping genes controlling hematocrit in the spontaneously hypertensive rat.

The genes that determine the baseline hematocrit level in humans and experimental animals are unknown. The spontaneously hypertensive rat (SHR), the most widely used animal model of human essential hypertension, exhibits an increased hematocrit when compared with the normotensive Brown Norway (BN-Lx) strain (0.54 +/- 0.02 vs. 0.44 +/- 0.02, p < 0.01). Distribution of hematocrit values among recombinant inbred (RI) strains derived from SHR and BN-Lx progenitors was continuous, which suggests a polygenic mode of inheritance. The narrow heritability of the hematocrit was estimated to be 0.32. The Eno2 marker on Chromosome (Chr) 4 showed the strongest association (p < 0.0001) with the observed variability of hematocrit among RI strains. The erythropoietin (Epo) gene, originally reported to be syntenic with Eno2, has been mapped to Chr 12, thus excluding it as a potential candidate gene for the increased hematocrit in the SHR. The current linkage data extend homologies between rat, mouse, and human chromosomes.

Genetic isolation of a region of chromosome 8 that exerts major effects on blood pressure and cardiac mass in the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) is the most widely studied animal model of essential hypertension. Despite > 30 yr of research, the primary genetic lesions responsible for hypertension in the SHR remain undefined. In this report, we describe the construction and hemodynamic characterization of a congenic strain of SHR (SHR-Lx) that carries a defined segment of chromosome 8 from a normotensive strain of Brown-Norway rats (BN-Lx strain). Transfer of this segment of chromosome 8 from the BN-Lx strain onto the SHR background resulted in substantial reductions in systolic and diastolic blood pressure and cardiac mass. Linkage and comparative mapping studies indicate that the transferred chromosome segment contains a number of candidate genes for hypertension, including genes encoding a brain dopamine receptor and a renal epithelial potassium channel. These findings demonstrate that BP regulatory gene(s) exist within the differential chromosome segment trapped in the SHR-Lx congenic strain and that this region of chromosome 8 plays a major role in the hypertension of SHR vs. BN-Lx rats.

A genetic linkage map of the rat derived from recombinant inbred strains.

We have constructed a genetic linkage map in the rat by analyzing the strain distribution patterns of 500 genetic markers in a large set of recombinant inbred strains derived from the spontaneously hypertensive rat and the Brown-Norway rat (HXB and BXH recombinant inbred strains). 454 of the markers could be assigned to specific chromosomes, and the amount of genome covered by the mapped markers was estimated to be 1151 centimorgans. By including a variety of morphologic, biochemical, immunogenetic, and molecular markers, the current map integrates and extends existing linkage data and should facilitate rat gene mapping and genetic studies of hypertension and other complex phenotypes of interest in the HXB and BXH recombinant inbred strains.

Effects of renin gene transfer on blood pressure and renin gene expression in a congenic strain of Dahl salt-resistant rats.

To investigate whether a BP-regulatory locus exists in the vicinity of the renin locus on rat chromosome 13, we transferred this chromosome segment from the Dahl salt-sensitive (S) rat onto the genetic background of the Dahl salt-resistant (R) rat. In congenic Dahl R rats carrying the S renin gene and fed an 8% salt diet, systolic BP was significantly lower than in progenitor Dahl R rats: 127 +/- 1 mmHg versus 138 +/- 4 mmHg, respectively (P < 0.05). Moreover, the decreased BP in the congenic Dahl R strain was associated with decreased kidney renin mRNA and decreased plasma renin concentration. These findings demonstrate that the Dahl S strain carries alleles in or near the renin locus that confer lower plasma renin concentration and lower BP than the corresponding alleles in the Dahl R strain, at least when studied on the genetic background of the Dahl R rat and in the environment of a high salt diet. The occurrence of coincident reductions in kidney renin mRNA, plasma renin concentration, and BP after interstrain transfer of naturally occurring renin gene variants strongly suggests that genetically determined variation in renin gene expression can affect BP.