Published online before print April 4, 2005, doi: 10.1161/01.HYP.0000161969.65767.0d
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2005;45:947.)
© 2005 American Heart Association, Inc.
Original Articles |
Klk1 as One of the Genes Contributing to Hypertension in Dahl Salt-Sensitive Rat
From the Research Institute, National Cardiovascular Center, Suita, Osaka, Japan.
Correspondence to Naoharu Iwai, MD, Department of Epidemiology, Research Institute, National Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail iwai{at}ri.ncvc.go.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A genome-wide quantitative trait loci analysis for blood pressure was performed using 107 male F2 rats derived from Dahl salt-sensitive and Lewis rats. Blood pressure was assessed by telemetry, and >400 microsatellite markers were used for genotyping. Two major quantitative trait loci for blood pressure were identified at chromosome 1 and chromosome 10. The expression levels of 366 transcripts around the chromosome 1 quantitative trait loci were assessed by RT-PCR, and we found that the Klk1 (kallikrein 1) and Ngfg (nerve growth factor gamma) mRNA levels were significantly reduced in the kidneys of Dahl salt-sensitive rats compared with those in Lewis rats. The expression levels of kallikrein 1 protein were also suppressed in Dahl salt-sensitive rats compared with those in Lewis rats. Because the kallikrein–kinin system has been shown to be involved in renal function, including salt homeostasis, it is likely that the reduced expression of Klk1 contributes to salt-sensitive hypertension in Dahl salt-sensitive rats.
Key Words: kallikreins • rats, Dahl • genetics
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dahl salt-sensitive rats have been widely used for the investigation of salt-sensitive hypertension. Sixteen genomic regions containing quantitative trait loci (QTLs) for blood pressure (BP) regulation have been reported in this strain.1–3 However, the genes responsible for this salt-sensitive hypertension have not yet been confirmed, despite numerous genetic studies.
A possible explanation for the lack of identification of the responsible genes in rat genetic studies to date might be the incompleteness of the rat genome data. The Rat Genome Project is now almost complete,4 and thus, it is now possible to obtain information on almost all of the protein-coding genes in candidate loci for a given phenotype.
Another drawback in previous rat genetic studies was the method used to assess BP. The BP levels of F2 rats were usually measured either with a tail-cuff method or with a temporary inserted arterial catheter. The validity of these classic methods of BP measurement is not concretely established, and furthermore, largely dependent on the skill levels of the individual researchers. Moreover, with these methodologies, circadian rhythm changes in BP cannot be adequately assessed. In the present study, these drawbacks were overcome by using telemetry.
In the present study, a genome-wide QTL analysis for BP was performed using 107 male F2 rats derived from Dahl salt-sensitive (DS) and Lewis (LEW) rats. The BP was measured by a telemetry system, and the genotypes of the F2 rats were determined with >400 genetic markers throughout the genome. Major QTLs were identified for daytime and nighttime BP (systolic and diastolic) levels. Next, we assessed the expression levels of 366 transcripts in the prominent QTLs and identified 2 transcripts that were differentially expressed in the kidneys of the 2 strains.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental Animals
DS and LEW rats were purchased from Sunplanet (Tokyo, Japan) and Charles River Japan (Yokohama, Japan), respectively. Rats were housed in a temperature-controlled room with the light on from 7:00 AM to 7:00 PM (daytime) and fed normal rat chow (0.5% NaCl; Clea Japan) and tap water ad libitum.
Male DS rats were mated with female LEW rats to produce F1 rats, and F1 rats were then intercrossed to produce an F2 population consisting of 107 male rats. F2 rats were started on an 8% NaCl diet (Oriental Yeast) at 5 weeks of age according to the protocol described by Rapp et al.1 Radiotelemetry devices (Data Sciences International) were implanted into the lower abdominal aorta of F2 rats at 9 weeks of age using sodium pentobarbital as an anesthetic agent (25 mg/kg IP). At 14 weeks of age, BP and heart rate were continuously measured for 44 hours (daytime 7:00 AM to 7:00 PM; nighttime 7:00 PM to 7:00 AM), and the data obtained during the latter 24 hours were used for analyses. The results were analyzed using Fluclet TM software (Dainippon Pharmaceutical).5 The present study was conducted in accordance with current guidelines for the care and use of experimental animals of the National Cardiovascular Center.
Genotyping and QTL Analysis
Genotyping was performed by PCR using appropriate PCR primer pairs (custom-made by Amersham Pharmacia Biotech), based on information from the Rat Genome Database (http://rgd.mcw.edu/) and Ratmap (http://ratmap.gen.gu.se/). We found sequence variations between DS and LEW rats in several genes, and these polymorphisms were also included in the genotyping data. The raw data are provided as a supplement (supplemental Table I, available online at http://hyper.ahajournals.org). We separately analyzed QTLs for daytime systolic BP (D-SBP), diastolic BP (D-DBP), nighttime systolic BP (N-SBP), and diastolic BP (N-DBP) levels using MapManager QTLb20.6 We first performed Quick Test to obtain significant thresholds for BP values. The likelihood ratio statistics (LRSs) for suggestive, significant, and highly significant loci were calculated to be 12.3, 17.7, and 25.9, respectively. Next, we performed marker regression for these 4 BP values. The most significant QTL was added to the background, and a second marker regression was performed to obtain the second QTL, which was added to the background to obtain the third QTL. We obtained 1 significant (chromosome 1) and 1 or 2 suggestive QTLs (chromosome 10 and chromosome 12) for these 4 BP values. Interval mapping of chromosome 1 was performed (free model) with other QTLs included as background.
RT-PCR Analysis of Expression Levels of Transcripts
For the screening of differentially expressed transcripts, we analyzed the expression levels of transcripts in the kidneys of DS and LEW rats (5 weeks of age) on a normal diet (n=2 in each group). The expression levels of transcripts were assessed by simple RT-PCR at 25 and 32 amplification stages because overamplification might obscure possible differences in expression levels. A 2.5-fold difference can be reliably detected by this method, provided the expression level of the target mRNA is between the Ngfg (nerve growth factor gamma) mRNA level and 1/1000 of that level (supplemental Figure I). After this initial screening, several possible transcripts that might be differentially expressed were subjected to precise estimation by competitive RT-PCR analyses with 18s ribosomal RNA as an internal control (QuantumRNA 18s Internal Standards Kit; Ambion). Because the 18s ribosome RNA level is extremely high, the PCR primers for 18s cDNA amplification are diluted by 18s competimers to reduce the efficiency of amplification to attain a comparable level to the target. The 18s competimers are modified at their 3' ends to block extension by DNA polymerase. The ratios of the 18s primers to 18s competimers were 3:7 for the Klk1 (kallikrein 1) and Ngfg mRNAs. The relative intensity of the PCR product from the Klk1 (721-bp) or Ngfg (529-bp) mRNA to the PCR product from 18s RNA (488-bp) was assessed by a densitometer. This assessment was performed within a range in which a linear relationship was maintained between the expression levels and the ratios of the PCR products. The expression levels were assessed in the kidneys of 5-week-old DS rats, 5-week-old LEW rats, 14-week-old DS rats under normal diet, 14-week-old LEW rats under normal diet, 14-week-old DS rats under a high-salt diet, and 14-week-old LEW rats under a high-salt diet (n=4 in each group).
The 366 transcripts that were analyzed and their amplification primers are provided as a supplement (supplemental Table II). Sense and antisense primers were derived from separate exons when the transcripts had introns. RNA samples were briefly treated with DNaseI and extracted with phenol-chloroform before reverse transcription to prevent possible amplification from contaminated genome DNA, which might obscure the expression levels of the intron-less transcript. Primers for the kallikrein gene family were derived from gene-specific sequence regions (Table 1) to avoid cross-amplification of homologous sequences, which had been confirmed by direct sequencing of the PCR products.
|
Western Blotting
Rabbit polyclonal antibody against rat urinary kallikrein was obtained from Merck Biosciences (formerly Calbiochem), which recognizes the active and inactive (prepro) forms.
LEW and DS rats (5 weeks old; n=4) were euthanized and kidneys were harvested. Kidneys were frozen in liquid N2 and were homogenized in Triton-based lysis buffer (1% Triton X-100, 20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerol phosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and a 0.1% protease inhibitor mixture [Nacarai Tesque]). The protein concentration was determined with the bicinchoninic acid method (Pierce). Equal amounts of proteins (60 µg) were separated by SDS-PAGE (7.5%) and transferred to a nitrocellulose membrane (BioTrace NT; Pall Corporation). After blocking with 5% BSA, membranes were incubated with primary antibody (1:1000 dilution) at 4°C overnight, and membrane-bound antibodies were visualized by horseradish peroxidase–conjugated secondary antibodies (1:10 000 dilution; 1 hour) and an ECL system (Amersham Biosciences). The expression levels were determined by densitometry.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Linkage to BP
A total of 418 polymorphic markers in all of the 107 F2 rats were genotyped, and some of them gave identical genotype data. Thus, the data on 383 effective genotypes were finally obtained (supplemental Table I).
Table 2 gives the results of the analysis using the MapManager QTX program for linkage to BP in the F2 (DSx LEW) population. Separate analyses were conducted of the D-SBP, D-DBP, N-SBP, and N-DBP values. Table 2 shows only the markers that yielded an LRS of at least a suggestive level of significance (ie, >12.3). The QTLs for N-SBP were around D1Rat27 and Pex12 (Ch10). Those for N-DBP were around D1Rat27 and D10Rat98, those for D-SBP were around D1Rat27, and those for D-DBP were around D1Rat27, Pex12, and D12Arb6.
|
Figure 1 provides LRS plots for the linkage to BP for chromosome 1 controlled by other suggestive loci. The chromosome 1 region near D1Rat27 is the most prominent and consistent QTLs for all of the BP values.
|
Identification of Candidate Genes in the Chromosome 1 Region
We next focused on the chromosome 1 region near D1Rat27 (at 90.3 mol/L). A total of 366 transcripts were found from this region between Cyp2a2 (82.1 mol/L) and loc292934 (99.5 mol/L), and RT-PCR expression analysis was performed (supplemental Table II). The expression of 240 genes was detected in the kidney of the 366 possible transcripts. As shown in Figure 2, the Klk1 and Ngfg mRNAs were found to be differentially expressed between the kidneys of DS and LEW rats. Klk1 and Ngfg are members of a large kallikrein gene family. Gene-specific primers for the kallikrein gene family were set up as described in Materials and Methods (Table 1). Direct sequencing confirmed that the PCR products originated from the Klk1 and Ngfg transcripts. Three-way ANOVA indicated that weeks of age (P=0.0023) and strain difference (P=0.0046) but not salt loading (P=0.8884) significantly affected the Klk1 mRNA levels (P=0.0006). The Klk1 mRNA level in LEW rats was 2.7-fold higher than that in DS rats. This differential expression was reconfirmed by the competitive RT-PCR method, in which a deletion mutant cRNA for Klk1 was used as an internal standard (supplemental Figure II).
|
Three-way ANOVA indicated that strain difference (P<0.0001) but not weeks of age (P=0.3629) or salt loading (P=0.3410) significantly affected the Ngfg mRNA levels (P=0.0005). The Ngfg mRNA level in LEW rats was 3.4-fold higher than that in DS rats at 5 weeks of age. The Ngfg mRNA level in LEW rats was 1.5-fold higher than that in DS rats at 14 weeks of age.
Sequence analysis of the entire Klk1 and Ngfg sequences including the 
1-kb promoter and 1-kb 3' regions in DS and LEW rats revealed no sequence difference except in the number of TC repeats in intron 1 of Ngfg: the number was 24 and 26 in DS and LEW rats, respectively.
This differential expression was also confimed by Western blot analysis (Figure 3). A primary antibody recognizing rat urinary kallikrein corresponding to KLK1 was used. In DS rats, KLK1 expression was significantly suppressed compared with LEW rats. It should be noted that preprokallikrein (44 kDa) and active kallikrein (38 kDa)7 were detected by this antibody, and the expression levels of both forms, especially those of active form, were suppressed in DS rats.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, a genome-wide F2 (DS X LEW) analysis with
400 markers and telemetry-based BP assessment was performed. The major QTLs for N-SBP, N-DBP, D-SBP, and D-DBP were identified. The most prominent and consistent QTLs for BP were identified on chromosome 1 near the kallikrein gene family. RT-PCR analyses showed that 2 transcripts in this region, namely Klk1 and Ngfg mRNA, were differentially expressed in the kidneys of the 2 strains. The kallikrein–kinin system has long been suspected to be involved in salt-sensitive hypertension in Dahl rats.8,9 Knockout mice that lack a bradykinin-B2 receptor have been shown to have salt-sensitive hypertension.10–12 A rat strain inbred for low urinary kallikrein excretion has also been reported to show salt-sensitive hypertension.13 As clarified in the present study, Klk1 mRNA expression levels in the kidney were lower in DS rats than in LEW rats. Therefore, it is likely that Klk1 contributes to hypertension in DS.
Lower urinary excretion of kallikrein-like activity has been reported in DS rats,8,9 and adenoviral transfer of the human kallikrein 1 gene has been reported to ameliorate hypertension and hypertension-associated target organ damage in DS rats.14,15 Based on these reports, it is likely that the low expression of Klk1 in DS rats is responsible for salt-sensitive hypertension. On the other hand, mice lacking Klk1 have been reported to exhibit cardiovascular abnormalities with normal BP.16,17 The hearts of these mice exhibited septum and posterior wall thinning and a tendency for dilatation, which led to decreased cardiac function. Thus, the normal BP in these mice, despite increased vascular resistance,17 reflects a reduced cardiac function. Therefore, this knockout model is not necessarily inconsistent with our hypothesis that the reduced Klk1 expression in DS rat kidneys is responsible for salt-sensitive hypertension in DS rats.
The LRS plots for BP values of Ch1 (Figure 1) are prominent and wide. Rapp et al reported the existence of 3 QTLs (QTL1a, QTL1b, and QTL2) on Ch1 on the basis of results in Dahl rat congenic strains with an introgressed LEW rat Ch1 segment.18 In these congenic experiments, the Klk1 locus seemed to have only a slight effect on BP (10mm Hg, congenic line (Ch1X3)X12; Figure 22), and an adjacent locus distal to the Klk1 locus appeared to be important (QTL1a). If 2 responsible genes reside in a very close range, the QTL plot will not give 2 separate peaks, but rather only 1 large pseudopeak. In such a case, it would not be possible to determine the precise location of the responsible genes only by linkage.
Thus, it is possible that the reduced expression of Klk1 in DS rats may have only modest effects on BP, and another gene near the Klk1 locus, which might have escaped the systematic expression analysis performed in the present study, might have profound effects on BP. Indeed, the expression analysis in the present study has several limitations; for example, subtle changes cannot be detected by the PCR-based expression analysis, the target tissue might not be the kidney, and the target gene might not be differentially expressed.
The unsolved problem of the present study is the lack of any significant sequence variations in Klk1 and Ngfg loci despite differential expression patterns. There would seem to be 2 possible explanations. One is that the regulation of Klk1 might be influenced by regulatory sequences acting over near-megabase distances. Recently, the existence of regulatory sequences acting over near-megabase distances has been suggested.19 The size of genomic regions functionally linked to a particular gene may thus need to be considerably expanded.19,20 The other explanation is that the downregulation of Klk1 in DS rats may be attributable to another gene near the Klk1 locus. The rat kallikrein gene family apparently consists of at least 22 genes encompassing >1 Mb.21–23 The physiological significance of each member in this family has not been determined. It is also possible that Klk1 expression may be modified to compensate for derangement in other members of this gene family. Such a compensatory decrease in Klk1 or Ngfg could lead to salt-sensitive hypertension in the Dahl rat.
Whatever the reason for the downregulation of Klk1 in DS rats, it has relevance to salt-sensitive hypertension because adenoviral transfer of human kallikrein 1 gene has been reported to ameliorate hypertension and hypertension-associated target organ damage in DS rats.14,15 Based on the latter hypothesis, Klk1 might not be a causative gene but might be an effector for salt-sensitive hypertension.
Ngfg is also a member of the kallikrein gene family, and the physiological substrates for Ngfg have not been confirmed.21–23 It is currently unclear whether the differential expression of Ngfg between the 2 strains might be related to salt-sensitive hypertension. Ngfg and Klk1 were found to be downregulated in DS, although no significant sequence difference was observed between DS and LEW rats. Because the existence of large-scale copy number polymorphism has been reported in the human genome,24 we assessed the copy number of the Klk1 locus in DS and LEW rats, and found no significant difference. Coordinate suppression of Klk1 and Ngfg might support either a compensation mechanism or the existence of long-range enhancers, as mentioned above.
In the present study, an assessment was made of 4 components of BP values: N-SBP, N-DBP, D-SBP, and D-DBP. Unexpectedly, the major QTLs for these traits were almost identical. Thus, the present results exclude the possibility that nighttime and daytime BP might be influenced by completely different sets of genes.
This investigation focused on the most prominent QTLs for BP levels (ie, those around the D1Rat27 locus). Although no assessment was made of the expression levels of transcripts around the second (Pex12 [peroxisomal biogenesis factor 12] at Ch10) or third (D12Arb6) QTL, we noted the existence of differentially expressed transcripts in the kidneys of DS and LEW rats, namely Pnpo (pyridoxine 5'-phosphate oxidase) at Ch10 and P2rx4 (purinergic receptor P2X, ligand-gated ion channel 4) at Ch12, the differences for which had been revealed by chance in our previous microarray analyses.25 However, a more thorough assessment will be necessary to identify candidate genes in these QTLs that could contribute to hypertension.
Garrett et al reported the existence of 9 BP QTLs (Ch1, 2, 3, 5, 8, 10, 16, 17, and 18) using F2 derived from DS X LEW, the same strains used in the present study. The most prominent BP QTLs were reported on Ch5 and 10. Of these 9 QTLs, the QTLs on Ch1, 5, 8, 10, and 17 were confirmed by the establishment of congenic strains.1,26,27 In the present study, BP QTLs were found on Ch1, 10, and 12. The most prominent BP QTL was on Ch1. Garrett et al measured BP by the tail-cuff method, and BP measured by this method is likely to be influenced by stress (eg, by heating of the tails and constrains). On the other hand, the BP QTLs detected by the telemetry system are free from such stress. The discrepancy observed between the 2 studies may be mainly attributable to the difference in the methods of BP measurement.
Perspectives
The Rat Genome Project is almost complete,4 and precise maps of protein-coding genes are now available. Furthermore, the rat is an appropriate animal for the physiological assessment of cardiovascular functions. Therefore, the rat is a suitable model animal for identifying genes that are related to cardiovascular diseases by means of a positional cloning strategy. On the other hand, the existence of various noncoding RNAs has been confirmed, including microRNAs, and it is highly probable that these noncoding RNAs are involved in gene regulation and gene–gene interactive network regulation.28,29 Moreover, the existence of large-scale copy number polymorphism in the human genome has also been reported.24 An expression analysis of just the protein-coding genes might not be sufficient for the identification of genes that contribute to salt-sensitive hypertension.
|
Acknowledgments |
|---|
The present study was supported by a grant-in-aid from the Salt Research Foundation 04C5 and the Program for the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.
Received October 28, 2004; first decision November 15, 2004; accepted February 28, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- Garrett MR, Dene H, Walder R, Zhang QY, Cicila GT, Assadnia S, Deng AY, Rapp JP. Genome scan and congenic strains for blood pressure QTL using Dahl salt-sensitive rats. Genome Res. 1998; 8: 711–723.
[Abstract/Free Full Text] - Garrett MR, Joe B, Dene H, Rapp JP. Identification of blood pressure quantitative trait loci that differentiate two hypertensive strains. J Hypertens. 2002; 20: 2399–2406.[CrossRef][Medline] [Order article via Infotrieve]
- Cicila GT, Rapp JP, Wang JM, St Lezin E, Ng SC, Kurtz TW. Linkage of 11 beta-hydroxylase mutations with altered steroid biosynthesis and blood pressure in the Dahl rat. Nat Genet. 1993; 3: 346–353.[CrossRef][Medline] [Order article via Infotrieve]
- Rat Genome Sequencing Project Consortium. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature. 2004; 428: 493–521.[CrossRef][Medline] [Order article via Infotrieve]
- Nagai R, Nagata S. New algorithmic-based digital filter processing system for real-time continuous blood pressure measurement and analysis in conscious rats. Comput Biol Med. 1995; 25: 483–494.[Medline] [Order article via Infotrieve]
- Manly KF, Cudmore RH Jr, Meer JM. Map manager QTX, cross-platform software for genetic mapping. Mamm Genome. 2001; 12: 930–932.[CrossRef][Medline] [Order article via Infotrieve]
- Takaoka M, Okamura H, Kuribayashi Y, Matsuoka H, Morimoto S. Activation of urinary inactive kallikrein by an extract from the rat kidney cortex. Life Sci. 1985; 16: 1015–1022.[CrossRef]
- Bouhnik J, Richoux JP, Huang H, Savoie F, Baussant T, Alhenc-Gelas F, Corvol P. Hypertension in Dahl salt-sensitive rats: biochemical and immunohistochemical studies. Clin Sci. 1992; 83: 13–22.[Medline] [Order article via Infotrieve]
- Churchill PC, Churchill MC, Bidani AK, Rabito SF. Kallikrein excretion in Dahl salt-sensitive and salt-resistant rats with native and transplanted kidneys. Am J Physiol. 1995; 269: F710–F717.[Medline] [Order article via Infotrieve]
- Alfie ME, Yang XP, Hess F, Carretero OA. Salt-sensitive hypertension in bradykinin B2 receptor knockout mice. Biochem Biophys Res Commun. 1996; 224: 625–630.[CrossRef][Medline] [Order article via Infotrieve]
- Alfie ME, Sigmon DH, Pomposiello SI, Carretero OA. Effect of high salt intake in mutant mice lacking bradykinin-B2 receptors. Hypertension. 1997; 29: 483–487.
[Abstract/Free Full Text] - Cervenka L, Harrison-Bernard LM, Dipp S, Primrose G, Imig JD, El-Dahr SS. Early onset salt-sensitive hypertension in bradykinin B2 receptor null mice. Hypertension. 1999; 34: 176–180.
[Abstract/Free Full Text] - Madeddu P, Varoni MV, Demontis MP, Chao J, Simson JA, Glorioso N, Anania V. Kallikrein-kinin system and blood pressure sensitivity to salt. Hypertension. 1997; 29: 471–477.
[Abstract/Free Full Text] - Chao J, Zhang JJ, Lin KF, Chao L. Human kallikrein gene delivery attenuates hypertension, cardiac hypertrophy, and renal injury in Dahl salt-sensitive rats. Hum Gene Ther. 1998; 9: 21–31.[Medline] [Order article via Infotrieve]
- Zhang JJ, Chao L, Chao J. Adenovirus-mediated kallikrein gene delivery reduces aortic thickening and stroke-induced death rate in Dahl salt-sensitive rats. Stroke. 1999; 30: 1925–1932.[Medline] [Order article via Infotrieve]
- Meneton P, Bloch-Faure M, Hagege AA, Ruetten H, Huang W, Bergaya S, Ceiler D, Gehring D, Martins I, Salmon G, Boulanger CM, Nussberger J, Crozatier B, Gasc JM, Heudes D, Bruneval P, Doetschman T, Menard J, Alhenc-Gelas F. Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice. Proc Natl Acad Sci U S A. 2001; 98: 2634–2639.
[Abstract/Free Full Text] - Bergaya S, Meneton P, Bloch-Faure M, Mathieu E, Alhenc-Gelas F, Levy BI, Boulanger CM. Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice. Circ Res. 2001; 88: 593–599.
[Abstract/Free Full Text] - Saad Y, Garrett MR, Rapp JP. Multiple blood pressure QTL on rat chromosome 1 defined by Dahl rat congenic strains. Physiol Genomics. 2001; 4: 201–214.
[Abstract/Free Full Text] - Nobrega MA, Ovcharenko I, Afzal V, Rubin EM. Scanning human gene deserts for long-range enhancers. Science. 2003; 302: 413.
[Free Full Text] - Flint J, Tufarelli C, Peden J, Clark K, Daniels RJ, Hardison R, Miller W, Philipsen S, Tan-Un KC, McMorrow T, Frampton J, Alter BP, Frischauf AM, Higgs DR. Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster. Hum Mol Genet. 2001; 10: 371–382.
[Abstract/Free Full Text] - Blaber M, Isackson PJ, Marsters JC Jr, Burnier JP, Bradshaw RA. Substrate specificities of growth factor associated kallikreins of the mouse submandibular gland. Biochemistry. 1989; 28: 7813–7819.[CrossRef][Medline] [Order article via Infotrieve]
- Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr Rev. 2001; 22: 184–204.
[Abstract/Free Full Text] - Fogaca SE, Melo RL, Pimenta DC, Hosoi K, Juliano L, Juliano MA. Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins. Biochem J. 2004; 380: 775–781.[CrossRef][Medline] [Order article via Infotrieve]
- Sebat J, Lakshmi B, Troge J, Alexander J, Young J, Lundin P, Maner S, Massa H, Walker M, Chi M, Navin N, Lucito R, Healy J, Hicks J, Ye K, Reiner A, Gilliam TC, Trask B, Oatterson N, Zetterberg A, Wigler M. Large-scale copy number polymorphism in the human genome. Science. 2004; 305: 525–528.
[Abstract/Free Full Text] - Okuda T, Sumiya T, Iwai N, Miyata T. Pyridoxine 5'-phosphate oxidase is a candidate gene responsible for hypertension in Dahl-S rats. Biochem Biophys Res Commun. 2004; 313: 647–653.[Medline] [Order article via Infotrieve]
- Ariyarajah A, Palijan A, Dutil J, Prithiviraj K, Deng Y, Deng AY. Dissecting quantitative trait loci into opposite blood pressure effects on Dahl rat chromosome 8 by congenic strains. J Hypertens. 2004; 22: 1495–1502.[CrossRef][Medline] [Order article via Infotrieve]
- Grondin M, Eliopoulos V, Lambert R, Deng Y, Ariyarajah A, Moujahidine M, Dutil J, Charron S, Deng AY. Complete and overlapping congenics proving the existence of a quantitative trait locus for blood pressure on Dahl rat Chromosome 17. Physiol Genomics. 2005 Jan 4. [Epub ahead of print]
- Morey C, Avner P. Employment opportunities for non-coding RNAs. FEBS Lett. 2004; 567: 27–34.[CrossRef][Medline] [Order article via Infotrieve]
- Ambros V. The functions of animal microRNAs. Nature. 2004; 431: 350–355.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  Y. Saad, S. Yerga-Woolwine, J. Saikumar, P. Farms, E. Manickavasagam, E. J. Toland, and B. Joe Congenic Interval Mapping of RNO10 Reveals a Complex Cluster of Closely-Linked Genetic Determinants of Blood Pressure Hypertension, November 1, 2007; 50(5): 891 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Y. Deng Positional Cloning of Quantitative Trait Loci for Blood Pressure: How Close Are We?: A Critical Perspective Hypertension, April 1, 2007; 49(4): 740 - 747. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||