The present study examined whether transfer of overlapping regions of chromosome 5 that include (4A+) or exclude the cytochrome P-450 (CYP) 4A genes from the Lewis rat alters the renal production of 20-hydroxyeicosatetraenoic acid (20-HETE) and/or the development of hypertension in congenic strains of Dahl salt-sensitive (S) rats. The expression of CYP4A protein and the production of 20-HETE in the renal outer medulla was greater in the 4A+ congenic strain than the levels seen in S rats or in overlapping control congenic strains that exclude the CYP4A region. Mean arterial pressure (MAP) rose from 122 +/- 2 to 190 +/- 7 mmHg in S rats and from 119 +/- 2 and 123 +/- 2 to 189 +/- 7 and 187 +/- 3 mmHg in the two control congenic strains fed an 8.0% NaCl diet for 3 wk. In contrast, MAP only increased from 112 +/- 2 to 150 +/- 5 mmHg in the 4A+ congenic strain. Chronic blockade of the formation of 20-HETE with N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide (TS-011; 1 mg/kg bid) restored the salt-sensitive phenotype in the 4A+ congenic strain and MAP rose to 181 +/- 6 mmHg after an 8.0% NaCl dietary challenge. TS-011 had no effect on the development of hypertension in S rats or the two control congenic strains. The pressure-natriuretic and diuretic responses were fivefold greater in the 4A+ congenic strain than in S rats. These results indicate that transfer of the region of chromosome 5 between markers D5Rat108 to D5Rat31 from the Lewis rat into the Dahl S genetic background increases the renal production of 20-HETE, improves pressure-natriuresis and opposes the development of salt-induced hypertension.

Previously, a blood pressure (BP) quantitative trait locus (QTL) on rat chromosome 9 (RNO9) was localized to a <2.4 cM interval using congenic strains generated by introgressing segments of RNO9 from the Dahl salt-resistant (R) rat into the background of the Dahl salt-sensitive (S) rat. Renal gene expression using Affymetrix gene chips was profiled on S and a congenic strain spanning the 2.4-cM BP QTL interval. This analysis identified 20 differentially expressed genes/expressed sequence tags. Of these, the locus with the greatest differential expression (30- to 35-fold) was regulated endocrine-specific protein 18 ( Resp18 ), which also mapped in the 2.4-cM BP QTL interval. Additional substitution mapping located the QTL to <0.4 cM or ≈493 kb. This newly defined QTL region still included Resp18 . Nucleotide variants were identified between S and R genomic DNA of Resp18 in the coding, 5′ regulatory and 3′ untranslated regions. The coding sequence variation (T/C) occurs in exon 2 and predicts an amino acid change (Ile/Val) in the protein product. Resp18 was considered a differentially expressed positional candidate for the QTL. To fine-map the BP QTL, we constructed a congenic strain with a smaller introgressed region. Compared with the S rat, this strain (1) had significantly lower BP, (2) did not contain the R form of Resp18 , and (3) did not retain the rather spectacular differential expression of Resp18. Together, these results demonstrate that a BP QTL independent of Resp18 exists within the newly defined 117-kb QTL region on RNO9.

To the Editor: Congenic rat strains are important tools for the genetic dissection of essential hypertension.1 Garrett et al recently demonstrated that a blood pressure (BP) quantitative trait locus (QTL) exists within a newly defined 117-kb QTL region on rat chromosome 9.2 By using microarray technology, the authors first found that the mRNA of Resp18 (endocrine-specific protein 18) is &7.27-fold lower in the kidney of S.R(9)x3A congenic rats (Dahl salt-resistant) than that of S rats (Dahl salt-sensitive). Furthermore, sequencing analysis revealed multiple mutations of Resp18 , particularly the sequence variation (T/C) in exon 2. However, a fine-map analysis showed that Resp18 is located just outside of the 117-kb QTL region; thus, Resp18 was eliminated as a candidate gene. The authors, however, did not address: (1) whether a homolog gene of Resp18 exists in genome, particularly in the 117-kb QTL region; (2) the significance of the Resp18 sequence variation (T/C) in exon 2; and (3) the possibilities that RESP18 plays a role in BP. We recently demonstrated that RESP18 not only shares significant sequence similarities with the N -terminal domain (amino acid 1&200) of IA-2, a dense-core vesicle (DCV)-transmembrane protein, but also shows a similar biological function to IA-2 in …

A blood pressure (BP) quantitative trait locus (QTL) was previously found on rat chromosome 9 using Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. A congenic strain, S.R(chr9), constructed by introgressing an R chromosomal segment into the S background, previously proved the existence of a BP QTL in a large 34.2-cM segment of chromosome 9. In the current work congenic substrains were constructed from the progenitor congenic strain, S.R(chr9). BP and heart weight comparisons between these congenic substrains and their S control localized the BP QTL to a 4.6-cM interval. Two solute carrier (Na(+)/H(+) exchanger) genes, Nhe2 and Nhe4, were excluded as candidates based on their map locations. A second iteration of congenic substrains was used to localize the QTL further to a 2.4-cM interval. Another solute carrier (Cl(-)/HCO3- exchanger) gene, Ae3, is in this reduced interval and was sequenced for both S and R strains, but no coding sequence variations were found. Ae3 mRNA was not differentially expressed in the kidney of congenic compared to S rats. Although the identity of the QTL remains unknown its map location has been reduced from an interval of 34.2 to 2.4 cM.

Objective To improve the localization of a blood pressure quantitative trait locus (BP QTL) on rat chromosome (RNO) 1. Methods Congenic substrains were derived from the progenitor congenic strains S.LEW(D1Mco4X1) and S.LEW(D1Mco4X5) which previously localized a BP QTL (region 2) to a 17cM interval on RNO1. The newly developed congenic substrains, along with control Dahl salt-sensitive (S) rats were fed a 2% NaCl diet for 24 days before their BP was compared by both tail-cuff and radiotelemetry methods. Results By comparing BP of these congenic substrains to that of S rats, we have refined the location of the BP QTL2 region to a 2.73 Mb genomic interval that contains 19 annotated genes in the latest rat genome assembly (version 2.1). Slc9a3, the gene encoding the Na+/H+ exchanger 3, originally a candidate gene in the BP QTL2 region, is excluded based on its map location. Conclusion Substitution mapping was used to reduce a BP QTL on RNO1 from 17 centimorgans (cM) to ∼1.4 cM (= 2.73 Mb). This region now contains 19 annotated rat candidate genes.

The Dahl salt-sensitive hypertensive (S) rat develops albuminuria early in life even on a low-salt diet. In contrast, the spontaneously hypertensive rat (SHR) is highly resistant to developing albuminuria despite elevated BP. An F(1) hybrid of S and SHR showed a low urinary albumin excretion (UAE) and low urinary protein excretion (UPE) similar to SHR, i.e., SHR was dominant. A genetic analysis was carried out on a large population (n = 276) obtained by backcrossing F(1) rats to the recessive S strain; the population was fed a low-salt diet. Genome scans done at 8, 12, and 16 wk of age yielded ten quantitative trait loci (QTL) for UAE and/or UPE with variable time-course patterns on nine rat chromosomes (RNO), i.e., RNO1, RNO2, RNO6, RNO8, RNO9, RNO10, RNO11, RNO13, and RNO19. There were two UPE QTL on RNO6. At most of the UAE and/or UPE QTL, the S allele was associated with increased excretion, except for one of the QTL on RNO6 and the QTL on RNO11, where the S allele caused decreased excretion. Only the UAE and UPE QTL on RNO10 co-localized with a BP QTL. The S allele on RNO10 caused higher BP and higher UAE. Two additional BP QTL were detected on RNO1 and RNO6. Most of the UAE and UPE QTL co-localized with QTL for kidney lesions characteristic of S rats. Multiple interactions were observed for UAE, many of which involved RNO2. In summary, UAE is highly polygenic and the majority of the QTL altering UAE do not co-localize with QTL for BP as evaluated by tail-cuff measurements of BP.

Previously we reported the construction of a congenic strain, S. LEW( 5), spanning a large region of rat chromosome 5. The Lewis (LEW) strain was the donor, and the Dahl salt-sensitive (S) strain was the recipient. The congenic strain included a blood pressure quantitative trait locus (QTL). In the present work, a series of nine congenic substrains were constructed from S. LEW( 5) which defined two closely linked blood pressure QTL in the region previously thought to contain only one. LEW low-blood-pressure alleles at both QTL were required for a major effect on blood pressure. Neither LEW allele alone had a significant effect on blood pressure. The two QTL were localized to regions 6.3 and 4.6 cM, and these were 1.0 cM apart.

OBJECTIVE:To describe genetic loci that differentiate blood pressures in two genetically hypertensive strains, the Dahl salt-sensitive (S) rat and the Albino Surgery (AS) rat.METHODS:A genome scan was performed using 222 genetic markers on an F2 population derived from two hypertensive strains, S and AS. The F2 rats were fed 8% NaCl for 5 weeks before blood pressure measurements were taken.RESULTS:Three blood pressure quantitative trait loci (QTL) were detected, one on each of rat chromosomes (RNO) 2, 4 and 8. The QTL on RNO4, unlike those on RNO2 and RNO8, was not detected in any of the previous seven linkage analyses reported with the S rat as one of the parental strains. Interactions between genetic loci throughout the genome were sought and interactions involving RNO4 with RNO8 and RNO4 with RNO14 were found. Including the new RNO4 locus identified in the present study, 16 distinct regions of the S rat genome have been demonstrated, by linkage analyses, to harbour loci that control blood pressure in the S rat.CONCLUSIONS:Increased blood pressure in two hypertensive strains, S and AS, is differentially regulated by genetic factors present on RNOs 2, 4 and 8. Therefore, of the 16 distinct genomic regions known to harbour blood pressure QTL in S rats, 13 are likely to contain blood pressure alleles that function similarly in the S rat and the AS rat, whereas three regions differentiate the two strains.

A quantitative trait locus (QTL) for blood pressure was previously detected on rat chromosome 10 (RNO10) by linkage analysis and confirmed by the construction of congenic strains that encompass large regions of RNO10. In the present study, the rat RNO10 blood pressure QTL was dissected by the further construction of congenic substrains. The original congenic region was shown to contain 2 blood pressure QTLs (QTL 1 and QTL 2) ≈24 cM apart. These were localized to a <2.6-cM region between markers D10Rat27 and D10Rat24 for QTL 1 and to a <3.2-cM region between D10Rat12 and D10Mco70 for QTL 2. Comparative mapping suggests that the rat RNO10 QTL 2 could be localized very close to a blood pressure QTL described by sib-pair analysis on human chromosome 17, but this is not definitively established because of multiple and complex chromosomal rearrangements between rodents and humans.

A series of congenic strains were constructed in which segments of chromosome (chr) 1 from Lewis (LEW) rats were introgressed into the Dahl salt-sensitive (S) strain. Three blood pressure quantitative trait loci (QTL) were defined. Two of these (QTL 1a and QTL 1b) were closely linked in the region between 1q31 and 1q35. The third blood pressure QTL (QTL region 2) was close to the centromere between 1p11 and 1q12, which includes the candidate gene Slc9a3 for sodium/hydrogen exchange. The blood pressure QTL 1a and QTL 1b defined here overlap significantly with QTL for disease phenotypes of renal failure, stroke, ventricular mass, and salt susceptibility defined in other rat strains, implying that these disease phenotypes and our blood pressure phenotype have causes in common. QTL 1b also corresponded approximately with a blood pressure QTL described on human chr 15. The QTL region 2 corresponded approximately with blood pressure QTL described on mouse chr 10 and human chr 6.

Blood pressure is a quantitative trait that has a strong genetic component in humans and rats. Several selectively bred strains of rats with divergent blood pressures serve as an animal model for genetic dissection of the causes of inherited hypertension. The goal is to identify the genetic loci controlling blood pressure, i.e., the so-called quantitative trait loci (QTL). The theoretical basis for such genetic dissection and recent progress in understanding genetic hypertension are reviewed. The usual paradigm is to produce segregating populations derived from a hypertensive and normotensive strain and to seek linkage of blood pressure to genetic markers using recently developed statistical techniques for QTL analysis. This has yielded candidate QTL regions on almost every rat chromosome, and also some interactions between QTL have been defined. These statistically defined QTL regions are much too large to practice positional cloning to identify the genes involved. Most investigators are, therefore, fine mapping the QTL using congenic strains to substitute small segments of chromosome from one strain into another. Although impressive progress has been made, this process is slow due to the extensive breeding that is required. At this point, no blood pressure QTL have met stringent criteria for identification, but this should be an attainable goal given the recently developed genomic resources for the rat. Similar experiments are ongoing to look for genes that influence cardiac hypertrophy, stroke, and renal failure and that are independent of the genes for hypertension.

53 P4504A1 and 4A2 genes are differentially expressed in the kidney and cosegregate with blood pressure in an F 2 cross of Dahl S and Lewis rats; however, it is unclear whether this pathway plays a causal or secondary role in the development of hypertension. The present study examined whether transfer of overlapping segments of chromosome 5 from D5Rat84 to D5Rat34 that excludes (4A- strain) or includes all four P4504A genes(4A+ strain),or just the 4A3 and 4A8 genes (4A3,8+ strain), of a Lewis rat alters blood pressure, proteinuria, and renal injury in 12 week old congenic strains of Dahl S rats fed a high salt diet (8% NaCl) for 3 weeks. MAP measured in conscious rats using a chronic catheter averaged 188 ± 3 mm Hg in male Dahl S rats (n=24), 190 ± 3 mm Hg in the 4A- congenic strain (n=24), 169 ± 4 mm Hg in the 4A+ congenic strain (n=30), and 172 ± 3 mm Hg in the 4A3,8+ strain (n=33). Transfer of the P4504A region also significantly reduced proteinuria, renal hypertrophy, and the degree of glomerular and tubulointerstitial injury. Protein excretion averaged 246 ± 22 mg/day in male Dahl S rats and 255 ± 25 mg/day, 129 ± 8 mg/day, and 169 ± 12 mg/day in the 4A-, 4A+, and 4A3,8+ strains, respectively. The antihypertensive and renoprotective effects of transfer of the P4504A region were associated with signficant increases in the expression of P4504A mRNA, P4504A protein levels, and the renal formation of 20-HETE. Interestingly, transfer of the P4504A region had no effect on blood pressure in female rats of the 4A+ or 4A3,8+ congenic strains, but it still reduced the degree of proteinuria and renal injury. These studies exclude the P4504A1 and 4A2 loci as the causal genes in this region in the development of hypertension in Dahl S rats. However, they indicate that the P4504A3 and 4A8 genes still lie in the interval between D5Rat108 and D5Rat54 that contains a locus that reduces blood pressure, is renoprotective, and alters the expression of P4504A genes in Dahl S rats.

Our purpose was to define quantitative trait loci (QTL) for blood pressure that differ between two widely used hypertensive rat strains, the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). A genome scan was done on an F-2 (S X SHR) population fed 8% NaCl for 4 wk. Three blood pressure QTL were detected, one on each of rat chromosomes (chr) 3, 8, and 9. For the chr 3 QTL the SHR allele increased blood pressure, and for chr 8 and 9 the S allele increased blood pressure. The QTL on chr 9 was exceptionally strong, having a LOD score of 7.3 and accounting for 30% of the phenotypic variance and a difference of 40 mmHg between homozygotes. A review of the literature in conjunction with the present data suggests that S and SHR are not different for the previously described prominent blood pressure QTL on chr 1, 2, 10, and 13. QTL for body weight on chr 4, 12, 18, and 20, each with an effect of about 30 g, were incidentally observed.

We performed an initial screen of 11 rat strains by use of a standard balloon injury to the left iliac artery to observe whether genetically determined differences existed in the development of neointimal hyperplasia. Neointimal hyperplasia was assayed 8 weeks after the vascular injury on coded microscopic sections. Statistically significant differences in the percentages of the vascular wall cross-sectional areas composed of intima (percentage intima) secondary to neointimal hyperplasia were noted among the different rat strains (P<0.02), with the Brown-Norway (BN), Dark Agouti, and Milan normotensive strain rats having the highest and the spontaneously hypertensive rats (SHR) having the lowest percentages of intima. In a separate experiment, F1 hybrids of SHRxBN strains and parental BN and SHR underwent the vascular injury, and the parental strains again showed a statistically significant difference from one another in the mean percentage of intima (P<0. 0001). The F1 hybrids showed an average percentage of intima intermediate between those of the parental strains. The average lumen size of the injured BN vessels were significantly smaller than that of the noninjured control vessels (P=0.044), but this significance disappeared when the circular areas of these vessels were calculated without taking neointimal growth into consideration (P=0.649). These results provide the groundwork for a genetic linkage analysis to identify the genes that influence the development of neointimal hyperplasia after vascular injury.

We previously reported that markers on rat chromosome 1 are genetically linked to blood pressure in an F-2 population derived from Dahl salt hypertension-sensitive (S) and Lewis (LEW) rats. Because there was evidence for more than one blood pressure quantitative trait locus (QTL) on chromosome 1, an initial congenic strain introgressing a large 118-centimorgan (cM) segment of LEW chromosome 1 into the S background had been constructed. This initial congenic strain had a reduced blood pressure compared with S rats, proving the existence of a blood pressure QTL, but not giving a good localization of the QTL. In the present work a series of five overlapping congenic substrains were produced from the original congenic strain in order to localize a blood pressure QTL to a 25-cM region near the center of chromosome 1. The congenic substrains also ruled out the Sa locus as a blood pressure QTL in the S vs. LEW comparison because the Sa locus was contained in a congenic substrain that did not alter blood pressure.