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Objectives: Ondansetron is an antagonist of 5-HT3 receptors mostly used as an antiemetic yet known to modulate metabolism and appetite. We tested the metabolic effects of ondansetron in newly derived congenic rat strain, carrying limited chromosome 8 regions of (PD) Brown Norway (BN) and polydactylous (PD) strain origins (including variant serotonin receptor Htr3b gene) within the genomic background of highly inbred model of metabolic syndrome, the spontaneously hypertensive rat (SHR)., Methods: Adult, standard diet-fed male rats of SHR and the congenic SHR.(PD/BN)8 strains received ondansetron (2mg/kg body weight/day) or vehicle (n=6/strain/treatment) via oral gavage for 14 days while we followed their metabolic and morphometric profiles including glucose tolerance and triacylgycerol and cholesterol concentrations in 20 lipoprotein fractions., Results: We fine-mapped the chromosome 8 differential segment in the new SHR.(PD/BN)8 congenic strain: it comprises BN-derived region together with an adjacent 422kb stretch of PD origin. The SHR.(PD/BN)8 rats were heavier than SHR, the fasting glucose was significantly higher in ondansetron-treated congenic than in SHR (post-hoc Tukey's HSD p=0.02). Compared to SHR, ondansetron induced significantly more robust increases of cholesterol and triacylglycerol concentrations in total, chylomicron, VLDL and HDL particles in the SHR.(PD/BN)8 congenic strain., Conclusion: We established new congenic model with distinct pharmacogenetic profile related to metabolic effects of ondansetron, facilitating thus the search for responsible genetic variants within the limited genomic region demarcated by the differential segment.

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

Aims: Therapeutic administration of retinoids is often accompanied with undesirable side effects, including an increase in lipid levels in up to 45% of treated patients. We tested the hypothesis of whether spontaneously hypertensive rat (SHR) and congenic SHR.PD-(D8Rat42-D8Arb23)/Cub (SHR-Lx) strains, differing only in a 14-gene region of chromosome 8 and previously shown to display differential sensitivity to the teratogenic effects of retinoic acid, could serve as a pharmacogenetic model set of the metabolic side effects of retinoid therapy., Materials & Methods: Male, 15-week old rats (n = 12/strain) of SHR and SHR-Lx strains were fed a high-sucrose diet for 2 weeks and subsequently treated either with all-trans retinoic acid (15 mg/kg) or only with a vehicle for 16 days (n = 6/strain/treatment), while still on the high-sucrose diet. We assessed the morphometric and metabolic profiles of all groups, including glucose tolerance tests, levels of insulin, adiponectin, free fatty acids, concentrations of triglycerides and cholesterol in 20 lipoprotein fractions under conditions of both high-sucrose diet and high-sucrose diet plus all-trans retinoic acid administration., Results & Conclusion: SHR-Lx displayed substantially greater sensitivity to a number of all-trans retinoic acid-induced metabolic dysregulations compared with SHR, resulting in impairment of glucose tolerance, increased visceral adiposity, and substantially greater increase of circulating triglyceride concentrations, accompanied by a shift towards their less favorable distribution into the lipoprotein fractions. These observations closely mimic the common side effects of retinoid therapy in humans, rendering SHR-Lx an experimental pharmacogenetic model of atRA-induced dyslipidemia.

Hypertension in humans and experimental models has a strong hereditary basis, but identification of causative genes remains challenging. Quantitative trait loci (QTLs) for hypertension and salt sensitivity have been reported on rat chromosome 18. We set out to genetically isolate and prioritize genes within the salt-sensitivity and hypertension QTLs on the spontaneously hypertensive rat (SHR) chromosome 18 by developing and characterizing a series of congenic strains derived from the SHR and normotensive Brown Norway rat strains. The SHR.BN-D18Rat113/D18Rat82 congenic strain exhibits significantly lower blood pressure and is salt resistant compared with the SHR. Transplantation of kidneys from SHR.BN-D18Rat113/D18Rat82 donors into SHR recipients is sufficient to attenuate increased blood pressure but not salt sensitivity. Derivation of congenic sublines allowed for the separation of salt sensitivity from hypertension QTL regions. Renal expression studies with microarray and Solexa-based sequencing in parental and congenic strains identified 4 differentially expressed genes within the hypertension QTL region, one of which is an unannotated transcript encoding a previously undescribed, small, nonprotein coding RNA. Sequencing selected biological candidate genes within the minimal congenic interval revealed a nonsynonymous variant in SHR transcription factor 4. The minimal congenic interval is syntenic to a region of human chromosome 18 where significant linkage to hypertension was observed in family based linkage studies. These congenic lines provide reagents for identifying causative genes that underlie the chromosome 18 SHR QTLs for hypertension and salt sensitivity. Candidate genes identified in these studies merit further investigation as potentially causative hypertension genes in SHR and human hypertension.

To identify renally expressed genes that influence risk for hypertension, we integrated expression quantitative trait locus (QTL) analysis of the kidney with genome-wide correlation analysis of renal expression profiles and blood pressure in recombinant inbred strains derived from the spontaneously hypertensive rat (SHR). This strategy, together with renal transplantation studies in SHR progenitor, transgenic and congenic strains, identified deficient renal expression of Cd36 encoding fatty acid translocase as a genetically determined risk factor for spontaneous hypertension.

Recombinant inbred (RI) strains (Prague HXB/BXH set) represent a unique model that allows for permanent summation of genetic and physiological information as well as the study of age-dependent changes in phenotypes and/or gene regulation. This study compared blood pressure (BP) measured in adult animals of RI strains by radiotelemetry with BP values obtained in conscious rats of comparable age subjected to short-term carotid catheterization or with those obtained by direct carotid puncture under ether anesthesia (almost 20 years ago). After radiotelemetry recording, the contribution of major vasoactive systems to BP maintenance was studied by consecutive inhibition of the renin-angiotensin system (RAS), sympathetic nervous system (SNS), and nitric oxide synthase. We found highly significant interrelationships among baseline BP values obtained by radiotelemetry, carotid catheterization, or carotid puncture. This indicates considerable stability of RI strains over the course of their long existence, and confirms the reliability of BP values used for genetic studies performed in the past. Subsequent analysis of vasoactive system participation revealed the importance of SNS for the maintenance of BP, as determined by either radiotelemetry or catheterization. The BP of catheterized rats also correlated closely with acute captopril-induced BP changes, but this was not the case for rats measured by radiotelemetry. NO-dependent vasodilatation matched the BP effects of SNS and RAS in both measuring conditions. Residual BP (recorded at sodium nitroprusside-induced dilatation of resistance vessels) was also responsible for a significant portion of the BP variation in RI strains. Our study confirms the validity of RI strains for the further genetic and physiological research of hypertension.

Approximately 30% of patients with hypertension have hepatic steatosis, and it has recently been proposed that fatty liver be considered a feature of the metabolic syndrome. Obesity, diet, and level of physical activity are likely factors modulating risk for hepatic steatosis, however genetic factors could also influence susceptibility or resistance to fatty liver in hypertensive or normotensive subjects. In genetic studies in spontaneously hypertensive rats (SHRs) and Brown Norway (BN) rats, we discovered that a variant form of sterol regulatory element binding transcription factor 1 (Srebf1 gene, SREBP-1 protein) underlies a quantitative trait locus (QTL) influencing hepatic cholesterol levels in response to a high cholesterol diet. Compared with the BN allele of Srebf1, the SHR allele of Srebf1 includes variants in the promoter and coding regions that are linked to hepatic deficiency of SREBP-1 mRNA and protein, reduced expression of the SREBP-1 target gene stearoyl-CoA desaturase 1, reduced promoter activity for SREBP-1c, and relative protection from dietary induced accumulation of liver cholesterol. Genetic correction of reduced SREBP-1 activity by derivation of congenic and transgenic strains of SHR increased hepatic cholesterol levels, thereby confirming Srebf1 as a QTL influencing hepatic lipid metabolism in the rat. The Srebf1 variant regulating hepatic cholesterol did not appear to affect blood pressure. These findings (1) are consistent with the results of association studies indicating that common polymorphisms affecting SREBP-1 may influence cholesterol synthesis in humans and (2) indicate that variation in Srebf1 may influence risk for hepatic steatosis.

We have developed a new, double-congenic rat strain BN-Lx.SHR2, which carries two distinct segments of chromosome 2 introgressed from the spontaneously hypertensive rat strain (SHR) into the genetic background of congenic strain BN-Lx, which was previously shown to express variety of metabolic syndrome features. In 16-wk-old male rats of BN-Lx and BN-Lx.SHR2 strains, we compared their glucose tolerance and triacylglycerol and cholesterol concentrations in 20 lipoprotein subfractions and the lipoprotein particle sizes under conditions of feeding standard and high-sucrose diets. Introgression of two distinct SHR-derived chromosome 2 segments resulted in decreased adiposity together with aggravation of glucose intolerance in the double-congenic strain. The BN-Lx.SHR2 rats were more sensitive to sucrose-induced rise in triacylglycerolemia. Although the total cholesterol concentrations of the two strains were comparable after the standard diet and even lower in BN-Lx.SHR2 after sucrose feeding, detailed analysis revealed that under both dietary conditions, the double-congenic strain had significantly higher cholesterol concentrations in low-density lipoprotein fractions and lower high-density lipoprotein fractions. We established a new inbred model showing dyslipidemia and mild glucose intolerance without obesity, attributable to specific genomic regions. For the first time, the chromosome 2 segments of SHR origin are shown to influence other than blood pressure-related features of metabolic syndrome or to be involved in relevant nutrigenomic interactions.

Identification of the genetic determinants of common diseases is a major challenge for current biomedical research. Combining linkage analyses of essentially monogenic cis-regulated expression phenotypes with oligogenic intermediate physiological phenotypes represents a promising approach for identification of quantitative trait loci at the molecular level. In the present review, a genetic analysis of cardiovascular phenotypes studied at several levels of complexity in rat recombinant inbred strains is described.

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.

The activity of the atrial natriuretic peptide receptor (Npr1) is altered in spontaneously hypertensive rats (SHR) in relation to its mRNA levels, suggesting abnormal transcriptional control in hypertension. A single-stranded conformational polymorphism caused by a repetitive dinucleotide segment of 10 TA in BN-Lx and of 40 TA in SHR was localized at position -943 relative to the transcription start site of the Npr1 gene, downstream of a putative cGMP-regulatory region, and was the only sequence difference noted between the two strains. Transient transfections of -1520 to -920 Npr1 promoter-SV40-luciferase fusion vector showed that the construct from BN-Lx stimulated the SV40 promoter, whereas that from SHR slightly inhibited it. In contrast to the BN-Lx construct, the activity of the SHR fragment was refractory to downregulation by atrial natriuretic peptide. Genotype-phenotype correlation studies in recombinant inbred strains (RIS) derived from BN-Lx and SHR crosses revealed significant correlations of the TA repeat with basal guanylyl cyclase activity and Npr1 mRNA levels. The correlations were heightened by a locus on chromosome 10 containing the Ace gene. The highest basal guanylyl cyclase activity and Npr1 mRNA values were found in RIS with both genes (Npr1/Ace) of BN genotypes, whereas the lowest were recorded in RIS, with the SHR genotypes at both loci. This was inversely correlated with diastolic blood pressure in these strains. In conclusion, the longer TA repeat unit in the promoter of Npr1 of SHR, in tandem with a putative cGMP responsive element, regulates the transcription of the Npr1 gene with consequences on diastolic blood pressure.

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.

It has recently been proposed that primary mutations in genes involved in fatty acid and lipid metabolism may contribute to the pathogenesis of insulin resistance and dyslipidemia often observed in spontaneous forms of hypertension. In the current study in the spontaneously hypertensive rat (SHR), we mapped and sequenced the gene encoding a key transcription factor known as ADD1 (adipocyte determination and differentiation factor 1) or SREBP-1c (sterol regulatory element binding protein- c) that has recently been identified as a master regulator of genes involved in the hepatic control of lipid and carbohydrate metabolism. We found that (1) the gene for ADD1/SREBP-1c maps to a region of rat Chromosome 10 previously reported to contain a quantitative trait locus involved in the regulation of hepatic cholesterol levels and (2) the SHR harbors a valine-to-methionine substitution in the COOH terminal portion of the ADD1/SREBP-1 protein that is not present in 44 other strains of laboratory rats. These findings, together with previous studies showing that transgenic expression of SREBP-1 isoforms has major effects on hepatic fatty acid and cholesterol biosynthesis, suggest that naturally occurring variation in the gene encoding the SREBP-1 isoforms might contribute to inherited variation in lipid metabolism in the SHR versus other strains of rats. These results should serve to motivate future transfection studies of the effect of the SHR mutant on SREBP-1 expression and activation in vitro, as well as the development of congenic and transgenic strains of SHR to investigate the effects of different variants of SREBP-1 on carbohydrate and lipid metabolism in vivo.

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.

Several groups including ours have demonstrated cardiac hyperplasia in neonates from genetically hypertensive rat strains. We have shown that similar problems exist in the kidney as well. More recently, we found that excessive heart and kidney weight is neonatally related to inhibition of apoptosis. Using recombinant inbred strains derived from a reciprocal cross between Brown Norway and spontaneously hypertensive rat progenitor strains, we mapped the inhibition of neonatal apoptosis to 2 distinct loci on chromosomes 1 (Myl 2) and 18 (Abrb 2). Positional candidate genes at these loci are being explored. These studies have also demonstrated that the loci determining kidney and heart weights in neonates are distinct from those determining increased organ weight in adults. The impact of blood pressure per se is also divergent because adult kidney weight is negatively correlated whereas heart weight is positively correlated with it. Analyses by extremes of low and high percentiles from fetal life to adulthood identified a single locus determining heart weight at Acaa on chromosome 8 in newborn (P=0.0003) and adult (P=0.016) rats. The Acaa region contains a DNA mismatch repair gene (hMLH1). The kinetics of neonatal growth through adulthood by prelabeling DNA with [(3)H]thymidine in pregnant mares showed that although the growth process is complex and nonlinear in the kidney of hypertensive rats, there is an increased turnover of cells, that is, reduced half-life of DNA. This observation is supported by the presence of shorter telomere fragments in kidneys of spontaneously hypertensive rats. These studies suggest that cardiovascular cells from hypertensive animals are subject to accelerated turnover, potentially leading to their accelerated aging.

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.

Stress is a critical contributor to cardiovascular diseases through its impact on blood pressure variability and cardiac function. Familial clustering of reactivity to stress has been demonstrated in human subjects, and some rodent models of hypertension are hyperresponsive to stress. Therefore, the present study was designed to uncover the genetic determinants of the stress response. We performed a total genome linkage search to identify the loci of the body temperature response to immobilization stress in a set of recombinant inbred strains (RIS) originating from reciprocal crosses of spontaneously hypertensive rats (SHR) with a normotensive Brown Norway Lx strain. Two quantitative trait loci (QTLs) were revealed on chromosomes (Chrs) 10 and 12 (logarithm of odds scores, 2.2 and 1. 3, respectively). The effects of these QTLs were enhanced by a high sodium diet (logarithm of odds scores, 4.0 and 3.3 for Chrs 10 and 12, respectively), which is suggestive of a salt-sensitive component for the phenotype. Congenics for Chr 10 confirmed both the QTL and the salt effect in RIS. Negatively associated loci were also identified on Chrs 8 and 11. Interaction between the loci of Chrs 10 and 12 was demonstrated, with the rat strains bearing SHR alleles at both loci having the highest thermal response to stress. Furthermore, the Y Chr of SHR origin enhanced the response to immobilization stress, as demonstrated in 2 independent models, RIS and Y Chr consomics. However, its full effect requires autosomes of the SHR strain. These findings provide the first evidence for the genetic determination of reactivity to stress with interactions between autosomal loci and between the Y and autosomal Chrs that contribute to the explanation of the 46% of variance in the stress response.

The SHR is the most widely studied animal model of hypertension. In this strain, as in many humans with essential hypertension, increased blood pressure has been reported to cluster with other risk factors for cardiovascular disease, including insulin resistance and dyslipidemia. However, the genetic mechanisms that mediate this clustering of risk factors for cardiovascular disease or the hypertension "metabolic syndrome" remain poorly understood. In the current studies, we have demonstrated (1) that a gene or genes responsible for a whole spectrum of cardiovascular risk factors mapped to a limited segment of the centromeric region of rat chromosome 4, (2) that a spontaneous deletion in the gene for Cd36 that encodes a fatty acid transporter and is located directly at the peak of QTL linkages on chromosome 4 has been indirectly linked to the transmission of insulin resistance, defective fatty acid metabolism, and increased blood pressure, and (3) based on complementation analysis in two transgenic lines expressing wild-type Cd36 on the genetic background of the SHR strain harboring the deletion variant of Cd36, we have established that defective Cd36 can be a determinant of disordered fatty acid metabolism, glucose intolerance, and insulin resistance 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.

Disorders of carbohydrate and lipid metabolism have been reported to cluster in patients with essential hypertension and in spontaneously hypertensive rats (SHRs). A deletion in the Cd36 gene on chromosome 4 has recently been implicated in defective carbohydrate and lipid metabolism in isolated adipocytes from SHRs. However, the role of Cd36 and chromosome 4 in the control of blood pressure and systemic cardiovascular risk factors in SHRs is unknown. In the SHR. BN-Il6/Npy congenic strain, we have found that transfer of a segment of chromosome 4 (including Cd36) from the Brown Norway (BN) rat onto the SHR background induces reductions in blood pressure and ameliorates dietary-induced glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. These results demonstrate that a single chromosome region can influence a broad spectrum of cardiovascular risk factors involved in the hypertension metabolic syndrome. However, analysis of Cd36 genotypes in the SHR and stroke-prone SHR strains indicates that the deletion variant of Cd36 was not critical to the initial selection for hypertension in the SHR model. Thus, the ability of chromosome 4 to influence multiple cardiovascular risk factors, including hypertension, may depend on linkage of Cd36 to other genes trapped within the differential segment of the SHR. BN-Il6/Npy strain.

It has been recently reported that increased hematocrit and hemoglobin values often accompany insulin resistance and compensatory hyperinsulinemia in humans. In the current study, we analyzed the relationship between hematocrit/hemoglobin on the one hand and insulin resistance, dyslipidemia, and hypertension on the other hand in HXB/BXH recombinant inbred (RI) strains derived from the spontaneously hypertensive rat (SHR) and the Brown Norway (BN) rat. The SHR progenitor strain had a significantly increased hematocrit values and it was also hypertensive and insulin-resistant when compared with the BN progenitor. The distribution of hematocrit and hemoglobin values among RI strains was continuous, suggesting a polygenic mode of inheritance. Analysis of RI strains revealed that hemoglobin was negatively correlated with insulin and insulin/glucose ratio, and that hematocrit was negatively correlated with insulin-stimulated glucose uptake in isolated adipocytes. There was no relationship between hematological parameters and blood pressure or lipid phenotypes in RI strains. The findings of the current study suggest that hematocrit and hemoglobin values might be added to the clustering variables related to the insulin resistance syndrome in the SHR strain.

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.

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.

It has been proposed that one of the primary events in the development of essential hypertension is a growth-related process initiated as early as during fetal development. Differences in kidney size have been observed between most rat models of hypertension and their respective controls. In this study, we analyzed relative kidney size (kidney weight/body wt) in a set of rat recombinant inbred strains (RIS) (N = 27) and their progenitors, the spontaneously hypertensive rat strain (SHR/Ola) and Brown Norway congenic strain (BN.1x), at two different ages, at birth and at 15 weeks. In the progenitors, the relative kidney weight was higher in the hypertensive than in the normotensive strain of both the newborn (P < 0.001) and adult (P < 0.001) animals. In the RIS, a significant correlation was found between the newborn and adult relative kidney weight (r = 0.49, P = 0.01), indicating that the two phenotypes share some of their genetic determinants. A total genome search of newborn and adult relative kidney weight was performed with a total of 453 genetic markers. These analyses revealed several suggestive quantitative trait loci (QTL), some of which were, indeed, significant for both newborn and adult relative kidney weight (such as, D3Mit9 on rat chromosome 3; r = -0.50, P < 0.01; r = -0.47, P < 0.01; respectively). Others, such as the locus on rat chromosome 1 (Rt6; r = -0.43, P < 0.05), were significant only for the adult relative kidney size. This QTL was found in close proximity to a region previously related to susceptibility to hypertensive renal disease in the fawn-hooded rat and, similarly to that study, its effect was found to be independent of blood pressure. Furthermore, a growth pattern of the kidneys after birth, evaluated as the difference between the newborn and adult relative kidney weight, was also subjected to total genome scan. Several suggestive QTL were identified. One of the most significant loci was found at the D1a marker on rat chromosome 17 (r = -0.51, P < 0.01), which was previously related to the determination of adult heart weight in the RIS. In conclusion, the current study demonstrates the usefulness of RIS in studies of hypertension-related phenotypes, some of which are abnormal before the development of high blood pressure. To better understand their role in the pathogenesis of hypertension, studies at different ages are needed, which are uniquely feasible in RIS.

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.

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.

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.

The frequent coincidence of hypertension and dyslipidemia suggests that related genetic factors might underlie these common risk factors for cardiovascular disease. To investigate whether quantitative trait loci (QTLs) regulating lipid levels map to chromosomes known to contain genes regulating blood pressure, we used a genome scanning approach to map QTLs influencing cholesterol and phospholipid phenotypes in a large set of recombinant inbred strains and in congenic strains derived from the spontaneously hypertensive rat and normotensive Brown-Norway (BN.Lx) rat fed normal and high cholesterol diets. QTLs regulating lipid phenotypes were mapped by scanning the genome with 534 genetic markers. A significant relationship (P < 0.00006) was found between basal HDL2 cholesterol levels and the D19Mit2 marker on chromosome 19. Analysis of congenic strains of spontaneously hypertensive rat indicated that QTLs regulating postdietary lipid phenotypes exist also on chromosomes 8 and 20. Previous studies in the recombinant inbred and congenic strains have demonstrated the presence of blood pressure regulatory genes in corresponding segments of chromosomes 8, 19, and 20. These findings provide support for the hypothesis that blood pressure and certain lipid subfractions can be modulated by linked genes or perhaps even the same genes.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of human essential hypertension. In the SHR strain, as in humans, the high blood pressure is determined multifactorially. Analysis of genetically segregating populations, derived from SHR and normotensive inbred strains, enabled localization of quantitative trait loci (QTLs) responsible for blood pressure regulation on several rat chromosomes. Analysis of specialized strains, congenic and recombinant inbred (RI) strains, helped to analyze some of these mapping results in detail: (1) analysis of congenic strains provided definitive evidence for the presence of blood pressure regulatory genes on chromosomes 8 and 13 and will enable mapping of responsible genes to limited segments of differential chromosomes, (2) the RI strains were shown to be especially useful for genome scanning studies of complex traits and for correlation analysis of blood pressure and other risk factors of cardiovascular disease such as cardiac hypertrophy, dyslipidemia, and insulin resistance.

Objective: To investigate whether mutations in the beta subunit of the epithelial sodium channel (Scnn1b) contribute to the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive rat., Design: We determined the chromosome location of the rat Scnn1b gene, tested for cosegregation with blood pressure, and sequenced near full-length Scnn1b complementary DNAs (cDNAs) from SHR and Dahl salt-sensitive rats., Methods: Chromosome mapping was performed by somatic cell hybrid analysis and by linkage analysis in recombinant inbred strains derived from SHR and Brown-Norway rats. Cosegregation analysis was performed by testing for correlations between blood pressure and Scnn1b genotypes in these strains. DNA sequencing was performed on cDNAs prepared from reverse-transcribed messenger RNA derived from rat kidney., Results: The Scnn1b gene was closely linked to the Sa gene on rat chromosome 1. Blood pressure correlated significantly with Scnn1b gene in the recombinant inbred strains. Analysis of near full-length Scnn1b cDNAs from SHR and Dahl rats failed to reveal any coding sequence mutations that could affect the predicted amino acid sequence of the Scnn1b protein., Conclusion: The Scnn1b gene maps near the Sa gene in a region of rat chromosome 1 involved in the inherited control of blood pressure. If disordered activity of the epithelial cell sodium channel contributes to the pathogenesis of hypertension in the SHR or Dahl models, it must stem from genetic lesions in sequences that regulate Scnn1b function or in sequences important to the structure or function of the other sodium channel subunits.

In the HXB and BXH recombinant inbred strains derived from the spontaneously hypertensive rat and the normotensive Brown Norway rat, we determined the strain distribution patterns of 500 genetic markers to scan the rodent genome for quantitative trait loci regulating cardiac mass and blood pressure. The markers spanned approximately 1,139 cM of the genome and were tested for correlations with left ventricular mass adjusted for body weight, and with systolic, diastolic, and mean arterial pressures. The marker for the dopamine 1A receptor (Drd1a) on chromosome 17 showed the strongest correlation with left ventricular heart weight (P = .00038, r = -0.59) and the relationship to heart weight was independent of blood pressure. The markers showing the strongest correlations with systolic, diastolic, and mean arterial pressure were D19Mit7 on chromosome 19 (P = .0012, r = .55), D2N35 on chromosome 2 (P = .0008, r = .56), and Il6 on chromosome 4 (P = .0018, r = .53), respectively. These studies demonstrate that the HXB and BXH strains can be effectively used for genome scanning studies of complex traits and have revealed several chromosome regions that may be involved in the genetic control of blood pressure and cardiac mass in the rat.

1. A significant portion of blood pressure variance is modified by the environment. 2. The present report summarizes evidence that: (i) the environmental response is genetically determined; (ii) various stressors can evoke a differential response in hypertensive animals and constitute its intermediate phenotypes; (iii) the response to heat stress can be assigned to a single 'thermosensitivity' locus; (iv) candidate genes of susceptibility to environmental stresses are member(s) of the heat stress gene (HSP) gene families; (v) a restriction fragment length polymorphism of hsp70 and a single base mutation in the 3'-untranslated region of hsp27 are associated with hypertension in recombinant inbred strains. 3. In conclusion, HSP gene variants may be causative in susceptibility to hypertension.

1. The HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway (BN.1x) rat, represent a very useful system for gene mapping and for genetic analysis of certain model diseases, such as spontaneous hypertension. 2. These RI strains were genotyped in multiple genetic polymorphisms and characterized in blood pressure and some intermediate phenotypes. 3. The analysis of RI strains has revealed that (i) a gene in the vicinity of the major histocompatibility complex (RT1) on chromosome 20, a kallikrein-related gene on chromosome 4 and the renin gene on chromosome 13 were significantly associated with blood pressure, and (ii) Na+ leak in red blood cells correlated with blood pressure whereas relative heart and kidney weights as well as platelet aggregation did not.

Objective: To determine whether the endothelin-1 or endothelin-3 genes are genetically linked with blood pressure and relative heart weight in segregating rat populations, in the context of an elevated dietary sodium chloride intake., Methods: Endothelin-1 and endothelin-3 genotypes of rats in segregating populations, derived from crosses of Dahl salt-sensitive (SS/Jr) rats with contrasting inbred strains, including Lewis rats, spontaneously hypertensive rats and Dahl salt-resistant (SR/Jr) rats, were determined using restriction fragment length polymorphisms. Segregating populations were fed a high (8%)-sodium chloride diet. Linkage of genotype with blood pressure or relative heart weight was determined by analysis of variance. Chromosomal location of the rat endothelin-3 gene was determined by genotyping a panel of recombinant inbred strains., Results: Two alleles for the endothelin-1 gene and three alleles for the endothelin-3 gene were identified. The endothelin-1 locus did not cosegregate with blood pressure or relative heart weight. The endothelin-3 locus cosegregated with blood pressure and relative heart weight in an SS/Jr x F1 (SS/Jr x SR/Jr) population, but not in populations containing a higher percentage of genes from the SR/Jr strain. The endothelin-3 and seminal vesicle protein-1 loci were linked and located on rat chromosome 3., Conclusion: The endothelin-3 gene is, or is linked to, a locus on chromosome 3 that regulates blood pressure and relative heart weight in inbred Dahl rats, and these effects were strongly dependent on the genetic background.

Thermosensitivity has been demonstrated in hypertensive rats and mice, and hypertension is frequent in hyperthermia-susceptible pigs. We have demonstrated that thermosensitivity segregates with hypertension in mice as a recessive trait in a single locus termed Tms. Since thermosensitivity can be demonstrated in cells obtained from neonatal hypertensive animals and persists after several passages in culture, it was of interest to study its cellular determinants. We undertook studies of candidate genes of cellular thermosensitivity, hsp70 being the major heat stress gene. First, we have observed an enhanced hsp70 mRNA accumulation in the hypertensive rat, mouse and human after heat shock of the whole animal, of its isolated organs or cultured cells. This increased accumulation of hsp70 mRNA in hypertension is due to its increased transcriptional rate. A restriction fragment length polymorphism (RFLP) of hsp70 was documented in hypertensive rats, which allowed the study of the segregation of this locus with hypertension in recombinant inbred strains of rats. At least one copy of hsp70 has been localized in RT1, the major histocompatibility complex of the rat, with polymorphism demonstrated with BamHI outside of the coding region of hsp70. This polymorphism segregates with 15 mmHg of systolic blood pressure. More recently, our studies led us to identify a polymorphism in another heat stress gene, hsp27. This polymorphism has been identified by PCR SSCP and is located in the 3' region, close to the termination translation codon.(ABSTRACT TRUNCATED AT 250 WORDS)

1. A structural alteration within the first intron of the renin gene in spontaneously hypertensive rats was demonstrated to co-segregate with blood pressure in some sets of F2 hybrids or recombinant inbred strains. There is no evidence as to whether restriction fragment length polymorphism of the renin gene is associated with any of the changes in the renin tissue level. For this reason we have determined renal renin activity in spontaneously hypertensive, Wistar-Kyoto and Brown Norway rats as well as in 22 recombinant inbred strains derived from F2 hybrids of spontaneously hypertensive and Brown Norway rats. 2. At the age of 4 months significantly lower renal renin activity was observed in spontaneously hypertensive rats than in both normotensive rat strains, Wistar-Kyoto and Brown Norway. The presence of the spontaneously hypertensive rat allele in recombinant inbred strains was associated with a substantially lower renal renin activity as compared with recombinant inbred strains bearing the Brown Norway rat allele. There was no relationship between renal renin activity and the polymorphism in either the angiotensinogen gene or the angiotensin-converting enzyme gene. 3. There was a borderline correlation between blood pressure and renal renin activity in recombinant inbred strains. Nevertheless, additional comparisons within recombinant inbred strains bearing the spontaneously hypertensive rat allele of the renin gene failed to reveal any significant relationship between blood pressure level and renal renin activity. 4. Our data suggest that the restriction fragment length polymorphism marking the renin gene of the spontaneously hypertensive rat is accompanied by an alteration in the renin-angiotensin system at the renal level.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: Hyper-responsive platelets are often found in essential hypertension. It has also been suggested that in hypertensive patients platelets may serve as an easily accessible indicator of abnormalities in contractile cell function. To test these suggestions, we analysed the relationship between platelet aggregation and genetic hypertension in the rat., Methods: Linear regression analysis of mean values of recombinant inbred strains was used to evaluate the relationship between blood pressure and ADP-induced platelet aggregation., Results: ADP-induced platelet aggregation in platelet-rich plasma in spontaneously hypertensive rats (SHR) was significantly decreased compared with in normotensive Brown Norway (BN) rats. However, in recombinant inbred strains, derived from (SHR x BN) F2 hybrids, correlation analysis revealed that platelet aggregation and blood pressure are independent traits., Conclusions: The present results suggest strongly that spontaneous hypertension and platelet hypo-aggregability in SHR were linked together by chance due to drift during selective inbreeding. The absence of a correlation between the two traits also indicates that alterations in platelet function in essential hypertension, often found in population-based studies, may have to be reaffirmed in genetically better-defined situations, e.g. by pedigree analysis.

Red blood cell Na+ content as well as ouabain-resistant Na+ and Rb+ (K+) transport (susceptible or resistant to inhibition by loop diuretics) were determined in spontaneously hypertensive rats (SHR) and normotensive Brown Norway (BN) rats the erythrocytes of which were incubated in either saline or Mg(2+)-sucrose medium. Elevated ouabain-resistant Na+ net uptake contrasted with slightly decreased red blood cell Na+ content in SHR compared with BN rats. Acceleration of furosemide- and bumetanide-sensitive Na+ fluxes contributed to enhanced ouabain-resistant Na+ influx into SHR erythrocytes in saline medium, whereas higher furosemide- or bumetanide-resistant Na+ efflux caused greater ouabain-resistant Na+ efflux in Mg(2+)-sucrose medium. Furosemide- and bumetanide-resistant Rb+ leaks were augmented in SHR erythrocytes. The association of the disclosed ion transport alterations with blood pressure was examined in 20 recombinant inbred strains derived from F2 SHR x BN hybrids. Ouabain-resistant Na+ uptake as well as furosemide- and bumetanide-resistant Na+ inward leaks (but not red blood cell Na+ content or furosemide- and bumetanide-sensitive Na+ net uptake) cosegregated with systolic and pulse pressures but not diastolic pressure of the recombinant inbred strains. In contrast, neither ouabain-resistant Na+ efflux nor any component of ouabain-resistant Rb+ uptake correlated positively with blood pressure of the recombinant inbred strains. Increased ouabain-resistant Na+ influx was compensated for by accelerated ouabain-sensitive Na+ extrusion because red blood cell Na+ content was not elevated in the hypertensive strains. Thus, high cell Na+ turnover rates might be related to genetic hypertension if an altered Na+ inward leak would be less effectively compensated for in tissues involved in cardiovascular regulation.

Previous studies from our laboratory have demonstrated that the intermediate phenotype of thermosensitivity is present in hypertensive mice and rats. Increased expression of hsp70 caused by increased transcription rate was demonstrated in vivo, in organs, and in cultured cells from spontaneously hypertensive rats and hypertensive mice. In this study, a polymorphism of this gene was revealed with BamHI enzyme by using a human hsp70 probe. A 4.4-kb fragment was visualized in normotensive rats (Brown-Norway BN.lx and Sprague-Dawley), and a 3.0-kb fragment was found in spontaneously hypertensive rats (SHR) of three different origins and in Wistar and Buffalo rats. Both fragments were present in the Wistar-Kyoto rat strain. The present study mapped the polymorphism of hsp70 into the RT1 complex in BN.1K and SHR.1N congenic strains. The hsp70 restriction fragment length polymorphism is associated with a blood pressure difference of 15 mm Hg in recombinant inbred strains. These results justify the search for a mechanism by which hsp70 could influence blood pressure.

The mode of blood pressure inheritance and some genetic markers of spontaneous hypertension were evaluated in a new set of recombinant inbred (RI) strains obtained by crossing of normotensive (BN.lx) and hypertensive (SHR) progenitor strains. Blood pressure values of RI strains were continuously distributed between both progenitor strains, although normotensive strains slightly prevailed. Statistical analysis suggested that there are three major genes and multiple minor genes responsible of the determination of spontaneous hypertension. The association between blood pressure and gene(s) within RT1 complex or gene(s) closely linked to it was found by RI strain analysis. This suggestion was confirmed by the detection of significant difference in blood pressure between SHR and SHR.1N congenic strains. Our results indicate that RT1 complex gene(s) may be involved in the development of high blood pressure.