Blood Pressure Quantitative Trait Loci Research Articles

Interaction Between Chromosome 2 and 3 Regulates Pulse Pressure in the Stroke-Prone Spontaneously Hypertensive Rat

In an F2 cross between stroke-prone spontaneously hypertensive (SHRSP) and Wistar Kyoto (WKY) rats, we previously identified blood pressure quantitative trait loci (QTL) on rat chromosome (RNO) 2 and a pulse pressure QTL on RNO3. The aims of this study were to confirm the QTL on RNO3 and to investigate interaction between RNO2 and RNO3 loci through the generation and phenotypic assessment of single RNO3 congenic (SP.WKY(Gla)3a) and bicongenic (SP.WKY(Gla)2a/3a) strains. Hemodynamic profiling, vascular function, and renal histology were examined in these newly generated strains along with the previously reported RNO2 congenic strain (SP.WKY(Gla)2a). Our results demonstrate significant equivalent reduction in systolic, diastolic, and pulse pressure phenotypes in SP.WKY(Gla)3a and SP.WKY(Gla)2a rats, whereas greater reductions were observed with the SP.WKY(Gla)2a/3a bicongenic strain achieving blood pressure levels similar to normotensive WKY rats. Epistasis was observed between pulse pressure QTL on RNO2 and 3 at baseline and during 1% salt challenge. Vascular function and renal pathology studies indicate that QTL on RNO3 are responsible for salt-induced kidney pathology, whereas QTL on RNO2 seem to have greater impact on vascular function. RNO3 congenic and bicongenic strains have confirmed the importance of SHRSP alleles in the RNO3 congenic interval on pulse pressure variability and end-organ damage. These strains will allow interrogation of complex gene-gene and gene-environment interactions contributing to salt-sensitive hypertension and renal pathology in the SHRSP rat.

Congenic Mapping and Sequence Analysis of the Renin Locus

Renin was the first blood pressure (BP) quantitative trait locus mapped by linkage analysis in the rat. Subsequent BP linkage and congenic studies capturing different portions of the renin region have returned conflicting results, suggesting that multiple interdependent BP loci may be residing in the chromosome 13 BP quantitative trait locus that includes Renin. We used SS-13(BN) congenic strains to map 2 BP loci in the Renin region (chr13: 45.2-49.0 Mb). We identified a 1.1-Mb protective Brown Norway region around Renin (chr13: 46.1-47.2 Mb) that significantly decreased BP by 32 mm Hg. The Renin protective BP locus was offset by an adjacent hypertensive locus (chr13: 47.2-49.0 Mb) that significantly increased BP by 29 mm Hg. Sequence analysis of the protective and hypertensive BP loci revealed 1433 and 2063 variants between Dahl salt-sensitive/Mcwi and Brown Norway rats, respectively. To further reduce the list of candidate variants, we regenotyped an overlapping SS-13(SR) congenic strain (S/renrr) with a previously reported BP phenotype. Sequence comparison among Dahl salt-sensitive, Dahl R, and Brown Norway reduced the number of candidate variants in the 2 BP loci by 42% for further study. Combined with previous studies, these data suggest that at least 4 BP loci reside within the 30-cM chromosome 13 BP quantitative trait locus that includes Renin.

Dahl (S x R) Congenic Strain Analysis Confirms and Defines a Chromosome 5 Female-Specific Blood Pressure Quantitative Trait Locus to <7 Mbp

The detection of multiple sex-specific blood pressure (BP) quantitative trait loci (QTLs) in independent total genome analyses of F2 (Dahl S x R)-intercross male and female rat cohorts confirms clinical observations of sex-specific disease cause and response to treatment among hypertensive patients, and mandate the identification of sex-specific hypertension genes/mechanisms. We developed and studied two congenic strains, S.R5A and S.R5B introgressing Dahl R-chromosome 5 segments into Dahl S chromosome 5 region spanning putative BP-f1 and BP-f2 QTLs. Radiotelemetric non-stressed 24-hour BP analysis at four weeks post-high salt diet (8% NaCl) challenge, identified only S.R5B congenic rats with lower SBP (−26.5 mmHg, P = 0.002), DBP (−23.7 mmHg, P = 0.004) and MAP (−25.1 mmHg, P = 0.002) compared with Dahl S female controls at four months of age confirming BP-f1 but not BP-f2 QTL on rat chromosome 5. The S.R5B congenic segment did not affect pulse pressure and relative heart weight indicating that the gene underlying BP-f1 does not influence arterial stiffness and cardiac hypertrophy. The results of our congenic analysis narrowed BP-f1 to chromosome 5 coordinates 134.9–141.5 Mbp setting up the basis for further fine mapping of BP-f1 and eventual identification of the specific gene variant accounting for BP-f1 effect on blood pressure.

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain l-2-hydroxy acid oxidase

Long chain l-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.

Mapping a novel blood pressure quantitative trait locus within a congenic strain spanning a single annotated gene containing segment on rat chromosome 10

By high resolution substitution mapping using alleles of the LEW rat introgressed onto the Dahl Salt‐sensitive (S) rat, a 320kb congenic segment was previously identified on rat chromosome 10 (RNO10:70750008‐71070581bp) that further increased the blood pressure (BP) of the S rat. A newer iteration of congenic substrains with introgressed segments ranging from 83kb (70750008‐70832849bp) to 134kb (70750008‐70884058bp) were constructed to further fine‐map this region. Out of these, the systolic BP of the congenic strain spanning a 134kb region was significantly lower compared to that of the S rat by −28 mm Hg (p<0.0001). Bioinformatics analysis of this critical 134kb congenic interval on the rat genome and comparative mapping using mouse and human genomes suggested a single protein coding gene, LOC688668 as the sole candidate gene for this locus. Detailed sequence and expression analyses of this gene will be presented. The significance of this work is that it represents a unique high resolution mapping project wherein the BP lowering effect of the 134kb congenic segment is independently observed when isolated from the longer 320kb congenic segment, which had an overall BP increasing effect.

Identification of Rtel1, Edn3, and Dnajc5 as candidate genes for rat chromosome 3 blood pressure Quantitative Trait Loci (QTLs) by gene expression profiling under multiple salt‐loading conditions

Previous work, comparing congenic rat strains carrying Dahl salt‐resistant (R) rat chromosome 3 (RNO3) alleles on a Dahl salt‐sensitive (S) rat background with the hypertensive S rats defined two blood pressure (BP) QTL‐containing intervals. Renal gene expression profiling of S.R(ET3×1) and S.R(ET3×2) congenic substrain rats, compared to S rats, was conducted in two microarray experiments using Affymetrix Rat RAE230 GeneChip sets. The first examined renal expression after 1) maintenance on a low dietary (0.4% NaCl) salt intake or 2) following acute (24 hour) exposure to an elevated dietary salt (4% NaCl) intake, identifying differentially expressed genes by two‐way analysis of variance. The second examined renal expression following chronic (28 day) exposure to an elevated dietary salt (2% NaCl) intake, identifying differential expression by Significance Analysis of Microarrays thresholding. Ingenuity Pathways Analysis (IPA) was used to highlight cellular molecular networks containing differentially expressed probesets likely altered among the three strains. Candidate genes were defined as mapping to RNO3 congenic regions and being 1) differentially‐expressed and/or 2) interacting with one or more genes in an IPA‐identified molecular network. Differential expression of Rtel1, Edn3, and Dnajc5 was found in both the acute and chronic NaCl‐loading experiments by qRT‐PCR.

Epistatic effects of multiple congenic regions on blood pressure and heart weight in Dahl rats under salt‐loading conditions

Mammalian complex traits are little studied for combinatorial gene‐gene interactions. To study epistasis among blood pressure (BP) quantitative trait loci (QTLs) we developed bi‐and tri‐congenic strains that introgress R‐rat congenic regions containing RNO3, RNO7, and RNO9 low BP alleles into S rats. S rats, RNO3+RNO7+RNO9 tri‐congenic, and RNO3+RNO7, RNO3+RNO9, and RNO7+RNO9 bi–congenic rats (n=16) were concomitantly raised on a low (0.4% NaCl) salt diet until 6 weeks‐old, fed a higher (2% NaCl) salt diet for 24 days, after which tail‐cuff BP, body weight (BW), and heart weight (HW) were measured. While all bi‐ and tri‐congenic strains had lower BP (P<0.0001) compared to the parental, S, strain, BP differences were not observed among the bi‐ and tri‐congenic strains. Thus, low BP alleles in the congenic regions were non‐additive under this salt‐loading condition. While bi‐ and tri‐congenic strains had significantly lower HW (P<0.0001) compared to S rats, only RNO3+RNO7+RNO9 and RNO3+RNO7 rats had lower BW‐adjusted HW (1201.8 and 1128.0 mg, respectively) compared to S rats (1267.2 mg; P=0.004 and P<0.0001, respectively). Thus, low BP QTL‐containing alleles showed non‐additive effects, while RNO3+RNO7 bicongenic rats having lower BW‐adjusted HW compared with RNO3+RNO7+RNO9 rats (P<0.002). These results provide evidence for strong interactions between previously identified BP QTLs.

Mapping a genetic biomarker of blood pressure to <807.3kb using two genetically hypertensive rats

Two of the most widely used strains for mapping quantitative trait loci (QTLs) for blood pressure (BP) are the Dahl Salt‐sensitive (S) rat and the Spontaneously Hypertensive Rat (SHR). To map a previously identified BP QTL that differentiate these two strains, we continued substitution mapping studies on a BP QTL on chromosome 9 identified by a linkage study. A total of 11 new congenic substrains were developed and characterized. BP and heart weights of two of these substrains with introgressed segments of 2.05Mb and 807.3Kb were both significantly (p<0.001) lower than that of the S by 56 ± 6.61 and 41 ± 8.55 mm Hg (p<0.001)), respectively. The mRNA and protein levels of Tmeff2 were lower in the congenic substrain spanning 2.05Mb. Further, because Tmeff2 was the only protein‐coding candidate gene within the substrain spanning 807.3kb, which also had a BP effect, it was prioritized as a candidate gene. Tmeff2 is known to be upregulated in patients from multiple cohorts with cardiac hypertrophy. Because Tmeff2 is also known to affect cell proliferation, we assessed the proliferation rates of cells obtained from S and the congenic substrains with BP effect. Proliferation of cardiac fibroblasts was significantly slower in the congenic compared with S (p<0.05). Taken together, these results suggest that variants within the 807.3Kb congenic segment that alter the expression of Tmeff2 can be prioritized as candidate biomarkers controlling blood pressure and associated cardiac hypertrophy in both rats and in humans.

Novel genes as primary triggers for polygenic hypertension

The discovery of causative genes leading to hypertension in animal models can reveal new mechanistic insights into blood pressure (BP) regulations. Previously, we isolated segments that harbor BP quantitative trait loci (QTLs) on rat chromosome 10 as defined by congenic strains made from crosses of inbred hypertensive Dahl salt-sensitive (DSS) and normotensive Lewis rats. The aim of the current study was to identify hypertension-causing genes for each QTL. Molecular analysis was performed. A systematic and comprehensive molecular analysis divulged particular genes that carry nonconserved mutations. Specifically, the proline rich 11 gene is likely responsible for C10QTL5. C10QTL1 is one of five genes, namely Benzodiazepine receptor associated protein 1, Loc689764, myotubularin related protein 4, protein phosphatase 1E, PP2C domain containing and ring finger protein 43. Loc100363423 with no known function is a candidate for C10QTL3. The ATP-binding cassette, subfamily A (ABC1), member 8a gene is probably responsible for C10QTL2. Primary genes initiating polygenic hypertension are those not known to be involved in BP modulation. Novel pathways towards BP homeostasis appear to underlie the functionality of C10QTL5, C10QTL1 and C10QTL3 and C10QTL2. Moreover, these genes may become innovative targets for the diagnosis and therapeutics of essential hypertension.

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats.

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.

Refined mapping of blood pressure quantitative trait loci using congenic strains developed from two genetically hypertensive rat models

Previously linkage and substitution mapping were conducted between the Dahl Salt-sensitive (S) rat and the Spontaneously Hypertensive Rat (SHR) to address the hypothesis that genetic contributions to blood pressure (BP) in two genetically hypertensive rat strains are different. Among the BP quantitative trait loci (QTLs) detected, two are located on chromosome 9 within large genomic segments. The goal of the current study was to develop new iterations of congenic substrains, to further resolve both of these BP QTLs on chromosome 9 as independent congenic segments. A total of 10 new congenic substrains were developed and characterized. The newly developed congenic substrains S.SHR(9)x8Ax11A and S.SHR(9)x10Ax1, with introgressed segments of 2.05 and 6.14 Mb, represented the shortest genomic segments. Both of these congenic substrains, S.SHR(9)x8Ax11A and S.SHR(9)x10Ax1 lowered BP of the S rat by 56 mm Hg (P<0.001) and 15 mm Hg (P<0.039), respectively. The BP measurements were corroborated by radiotelemetry. Urinary protein excretion was significantly lowered by SHR alleles within S.SHR(9)x10Ax1 but not by S.SHR(9)x8Ax11A. The shorter of the two congenic segments, 2.05 Mb was further characterized and found to contain a single differentially expressed protein-coding gene, Tomoregulin-2 (Tmeff2). The protein expression of Tmeff2 was higher in the S rat compared with S.SHR(9)x8Ax11A, which also had lower cardiac hypertrophy as measured by echocardiography. Tmeff2 is known to be upregulated in patients from multiple cohorts with cardiac hypertrophy. Taken together, Tmeff2 can be prioritized as a candidate gene for hypertension and associated cardiac hypertrophy in both rats and in humans.

Global identification of the genes and pathways differentially expressed in hypothalamus in early and established neurogenic hypertension

The hypothalamus has an important etiological role in the onset and maintenance of hypertension and stress responses in the Schlager high blood pressure (BP) (BPH/2J) mouse, a genetic model of neurogenic hypertension. Using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays we identified 1,019 hypothalamic genes whose expression differed between 6 wk old BPH/2J and normal BP (BPN/3J) strains, and 466 for 26 wk old mice. Of these, 459 were in 21 mouse BP quantitative trait loci. We validated 46 genes by qPCR. Gene changes that would increase sympathetic outflow at both ages were: Dynll1 encoding dynein light chain LC8-type 1, which physically destabilizes neuronal nitric oxide synthase, decreasing neuronal nitric oxide, and Hcrt encoding hypocretin and Npsr1 encoding neuropeptide S receptor 1, each involved in sympathetic response to stress. At both ages we identified genes for inflammation, such as CC-chemokine ligand 19 (Ccl19), and oxidative stress. Via reactive oxygen species generation, these could contribute to oxidative damage. Other genes identified could be responding to such perturbations. Atp2b1, the major gene from genome-wide association studies of BP variation, was underexpressed in the early phase. Comparison of profiles of young and adult BPH/2J mice, after adjusting for maturation genes, pointed to the proopiomelanocortin-α gene (Pomc) and neuropeptide Y gene (Npy), among others, as potentially causative. The present study has identified a diversity of genes and possible mechanisms involved in hypertension etiology and maintenance in the hypothalamus of BPH/2J mice, highlighting both common and divergent processes in each phase of the condition.

Systematic polymorphism discovery after genome-wide identification of potential susceptibility loci in a hereditary rodent model of human hypertension

Genetic strategies such as linkage analysis and quantitative trait locus (QTL) mapping have identified a multitude of loci implicated in the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR). While several candidate genetic regions have been identified in the SHR and its control, the Wistar–Kyoto rat (WKY), systematic follow-up of candidate identification with polymorphism discovery has not been widespread. In the current report, we develop a data-mining strategy to identify candidate genes for hypertension in the SHR, and then sequence each gene in the SHR and WKY strains. We integrate blood pressure QTL data, microarray data and data-mining methods. First, we determined the set of genes differentially expressed in SHR and WKY adrenal glands. Next, the chromosomal position of all differentially expressed genes was compared with peak marker position of all reported SHR blood pressure QTLs. We also identified the set of differentially expressed genes with the most extreme fold-change. Finally, the QTL positional candidates and the genes with extreme differential expression were proposed as candidate genes if they had biologically plausible roles in hypertensive pathology. We identified seven candidate genes that merit resequencing (catechol-O-methyltransferase [Comt], chromogranin A [Chga], dopamine beta-hydroxylase [Dbh], electron transferring flavoprotein dehydrogenase [Etfdh], endothelin receptor type B [Ednrb], neuropeptide Y [Npy] and phenylethanolamine-N-methyltransferase [Pnmt]), and then discovered polymorphism in four of these seven candidate genes. Chga is proposed as the strongest candidate for additional functional investigation. Our method for candidate gene identification is portable and can be applied to microarray data from any tissue, in any disease model with a QTL database.

Normotension in Lewis and Dahl salt-resistant rats is governed by different genes

Inbred rodent models simulating essential hypertension and normotension are useful tools in discovering genes controlling blood pressure (BP) homeostasis. An analysis of a F2 population made from crosses of hypertensive Dahl salt-sensitive (DSS) and normotensive Lewis rats did not detect a BP quantitative trait locus (QTL) on chromosome 7 (Chr 7). However, false negativity could not be excluded. If a BP QTL could be proven to exist, what gene(s) may be responsible for this QTL. We first constructed reciprocal congenic strains for a Chr 7 segment and determined functional domains of prominent candidate genes. A congenic strain made in the DSS rat background exhibited a BP effect, indicating that a BP QTL, C7QTL, inhabits Chr 7. Contrarily, a congenic strain constructed in the Lewis rat background did not change BP, demonstrating a dependence of C7QTL on the DSS rats environment. Among the candidate genes, tachykinin 2 (Tac2), neurexophilin 4 (Nxph4) and retinol dehydrogenase 2 (Rdh2) bear nonsynonymous changes comparing DSS and Lewis rats, but are the same comparing DSS and Dahl salt-resistant (DSR) rats. In contrast, the Lewis alleles of 11-beta-hydroxylase (Cyp11b1), aldosterone synthase (Cyp11b2) and Cytochrome P-450 11B3 (Cyp11b3) are identical to those of DSS rats, but different from those of DSR rats. Thus, the failure to detect a linkage between a Chr 7 segment and BP in F2(DSS × Lewis) can be attributed to false negativity. Tac2, Nxph4 and Rdh2 are priority candidate genes for C7QTL. Lewis and DSR rats are both normotensive, but their underlying genetic determinants are different.

Augmented Rififylin Is a Risk Factor Linked to Aberrant Cardiomyocyte Function, Short-QT Interval and Hypertension

Using congenic strains of the Dahl salt-sensitive (S) rat introgressed with genomic segments from the normotensive Lewis rat, a blood pressure quantitative trait locus was previously mapped within 104 kb on chromosome 10. The goal of the current study was to conduct extensive phenotypic studies and to further fine-map this locus. At 14 weeks of age, the blood pressure of the congenic rats fed a low-salt diet was significantly higher by 47 mm Hg (P<0.001) compared with that of the S rat. A time-course study showed that the blood pressure effect was significant from very young ages of 50 to 52 days (13 mm Hg; P<0.01). The congenic strain implanted with electrocardiography transmitters demonstrated shorter-QT intervals and increased heart rate compared with S rats (P<0.01). The average survival of the congenic strain was shorter (134 days) compared with the S rat (175 days; P<0.0007). The critical region was narrowed to <42.5 kb containing 171 variants and a single gene, rififylin. Both the mRNA and protein levels of rififylin were significantly higher in the hearts of the congenic strain. Overexpression of rififylin is known to delay endocytic recycling. Endocytic recycling of fluorescently labeled holotransferrin from cardiomyocytes of the congenic strain was slower than that of S rats (P<0.01). Frequency of cardiomyocyte beats in the congenic strain (62±9 bpm) was significantly higher than that of the S rat (24±6 bpm; P<0.001). Taken together, our study provides evidence to suggest that early perturbations in endocytic recycling caused by the overexpression of Rffl is a novel physiological mechanism potentially underlying the development of hypertension.

Defining a rat blood pressure quantitative trait locus to a

Evidence from multiple linkage and genome-wide association studies suggest that human chromosome 2 (HSA2) contains alleles that influence blood pressure (BP). Homologous to a large segment of HSA2 is rat chromosome 9 (RNO9), to which a BP quantitative trait locus (QTL) was previously mapped. The objective of the current study was to further resolve this BP QTL. Eleven congenic strains with introgressed segments spanning <81.8 kb to <1.33 Mb were developed by introgressing genomic segments of RNO9 from the Dahl salt-resistant (R) rat onto the genome of the Dahl salt-sensitive (S) rat and tested for BP. The congenic strain with the shortest introgressed segment spanning <81.8 kb significantly lowered BP of the hypertensive S rat by 25 mmHg and significantly increased its mean survival by 45 days. In contrast, two other congenic strains had increased BP compared with the S. We focused on the <81.8 kb congenic strain, which represents the shortest genomic segment to which a BP QTL has been mapped to date in any species. Sequencing of this entire region in both S and R rats detected 563 variants. The region did not contain any known or predicted rat protein coding genes. Furthermore, a whole genome renal transcriptome analysis between S and the <81.8 kb S.R congenic strain revealed alterations in several critical genes implicated in renal homeostasis. Taken together, our results provide the basis for future studies to examine the relationship between the candidate variants within the QTL region and the renal differentially expressed genes as potential causal mechanisms for BP regulation.

Positional mapping of a rat blood pressure QTL to a congenic segment spanning &lt;82kb with no rat gene predictions

Genome‐wide association studies in humans suggest that a large number of alleles, each without a major effect, contribute to the genetics of blood pressure (BP). Our laboratory is focused on establishing a cause‐effect relationship between alleles and BP using rat genetic models of hypertension. Previously, a BP quantitative trait locus (QTL) was mapped within a congenic strain of the Dahl salt‐sensitive (S) rat containing 1.41Mb of rat chromosome 9 (RNO9) introgressed from the Dahl salt‐resistant (R) rat. High resolution mapping was conducted with this congenic strain as the progenitor. Eleven substrains with introgressed segments from 81.8kb to 1.33Mb were developed and tested for BP. The congenic strain with the shortest introgressed segment, S.R(9)INHx3x2Bx1x8 spanning 81.8kb had a significant BP lowering effect (−25, p&lt;0.001) compared to the S rat. The BP of S.R(9)INHx3x2Bx1x8 was further confirmed by both telemetry and tail‐cuff methods. The mean survival of S.R(9)INHx3x2Bx1x8 congenic rats was longer than that of the S by 44 days. Sequencing of the 81.8kb region revealed 371 DNA variants. The 81.8kb segment does not contain any known rat genes. The critical region contains 658bp which is 99.39% identical to a genomic segment on rat chromosome 2 and contains 4 SNPs. Transcripts and proteins encoded by the gene Tns1 located at the 3′ end of this genomic segment on RNO9 and the gene Nup155 located at the 3′ end of the identical genomic segment on RNO2 were both expressed lower in S compared to S.R(INHx3x2Bx1x8). This data suggests that a common regulatory element of Tns1 and Nup155 located within S.R (INHx3x2Bx1x8) could be a candidate BP QTL.

Blood pressure-independent factors determine the susceptibility to delayed neuronal death in the stroke-prone spontaneously hypertensive rats.

The stroke-prone spontaneously hypertensive rat (SHRSP) is vulnerable to delayed neuronal death (DND) in the CA1 subfield of the hippocampus after the transient forebrain ischemia by the occlusion of the bilateral carotid arteries. The present study was designed to show that the genetic factors independent of high blood pressure contributed to the high incidence of DND in SHRSP. Male rats of the four strains, SHRSP/Izm, SHRSP/Ngsk, SHR/Izm and a congenic strain for the blood pressure quantitative trait locus on chromosome 1 [SHRSP.WKY-(D1Wox29-D1Arb21)/Izm]were used in the experiments. At 13 weeks of age, the bilateral carotid arteries of rats were occluded for 10 min under anesthesia with their body temperature kept at 37 degreeC. Seven days after the transient ischemia, the loss of the pyramidal cells in the CA1 was evaluated histologically. In some experiments, the blood flow was monitored with a laser Doppler flowmeter during the transient ischemia. The blood pressure in SHRSP/Izm was significantly greater than that in the other three strains. The incidence of DND, however, was not significantly different among SHRSP/Izm, SHRSP/Ngsk and the congenic strain (82, 74 and 65%, respectively), while SHR/Izm showed a significantly lower incidence (20 percent). Neither a significant correlation between the incidence of DND and the blood flow reduction during the occlusion, nor a significant inter-strain difference in the blood flow reduction was observed. The genetic factors independent of high blood pressure may contribute to the greater susceptibility to DND in SHRSP.

Genetic Analysis of Blood Pressure in 8 Mouse Intercross Populations

The genetic basis of hypertension is well established, yet very few genes that cause common forms of hypertension are known. Quantitative trait locus (QTL) analyses in rodent models can guide the search for human hypertension genes, but the excellent genetic resources for mice have been underused in this regard. To address this issue, we surveyed blood pressure variation in mice from 37 inbred strains and generated 2577 mice in 8 intercross populations to perform QTL analyses of blood pressure. We identified 14 blood pressure QTL in these populations, including > or =7 regions of the mouse genome not linked previously to blood pressure. Many QTL were detected in multiple crosses, either within our study or in studies published previously, which facilitates the use of bioinformatics methods to narrow the QTL and focus the search for candidate genes. The regions of the human genome that correspond to all but 1 of the 14 blood pressure QTL in mice are linked to blood pressure in humans, suggesting that these regions contain causal genes with a conserved role in blood pressure control. These results greatly expand our knowledge of the genomic regions underlying blood pressure regulation in mice and support future studies to identify the causal genes within these QTL intervals.

Positional identification of variants of Adamts16 linked to inherited hypertension

A previously reported blood pressure (BP) quantitative trait locus on rat Chromosome 1 was isolated in a short congenic segment spanning 804.6 kb. The 804.6 kb region contained only two genes, LOC306664 and LOC306665. LOC306664 is predicted to translate into A Disintegrin-like and Metalloproteinase with Thrombospondin Motifs-16 (Adamts16). LOC306665 is a novel gene. All predicted exons of both LOC306664 and LOC306665 were sequenced. Non-synonymous variants were identified in only one of these genes, LOC306664. These variants were naturally existing polymorphisms among inbred, outbred and wild rats. The full-length rat transcript of Adamts16 was detected in multiple tissues. Similar to ADAMTS16 in humans, expression of Adamts16 was prominent in the kidney. Renal transcriptome analysis suggested that a network of genes related to BP was differential between congenic and S rats. These genes were also differentially expressed between kidney cell lines with or without knock-down of Adamts16. Adamts16 is conserved between rats and humans. It is a candidate gene within the homologous region on human Chromosome 5, which is linked to systolic and diastolic BP in the Quebec Family Study. Multiple variants, including an Ala to Pro variant in codon 90 (rs2086310) of human ADAMTS16, were associated with human resting systolic BP (SBP). Replication study in GenNet confirmed the association of two variants of ADAMTS16 with SBP, including rs2086310. Overall, our report represents a high resolution positional cloning and translational study for Adamts16 as a candidate gene controlling BP.