Published online before print October 15, 2007, doi: 10.1161/HYPERTENSIONAHA.107.095349
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(Hypertension. 2007;50:1134.)
© 2007 American Heart Association, Inc.
Original Articles |
Candidate Genes That Determine Response to Salt in the Stroke-Prone Spontaneously Hypertensive Rat
Congenic Analysis
From the BHF Glasgow Cardiovascular Research Centre (D.G., M.W.Mc.B., M.G., K.G., E.B., W.H.M., J.D.McC., J.M.P., A.F.D.) University of Glasgow, Glasgow, United Kingdom, and the Kidney Research Centre (A.M., R.M.T.), University of Ottawa, Ottawa Health Research Institute, Ottawa, Ontario, Canada.
Correspondence to Anna F. Dominiczak, BHF Glasgow Cardiovascular Research Centre, 126 University Pl, Glasgow, G12 8TA United Kingdom. E-mail dg74s{at}clinmed.gla.ac.uk
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Abstract |
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The existence of blood pressure quantitative trait loci exaggerated by salt on rat chromosome 2 has been confirmed previously using congenic strains derived from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. This study aimed to dissect the implicated chromosome 2 region and to identify candidate genes based on microarray expression profiling and real-time PCR. A marker-assisted breeding strategy generated congenic strains SP.WKYGla2a (D2Rat13-D2Rat157), SP.WKYGla2c* (D2Wox9-D2Mgh12), and SP.WKYGla2k (D2Mit21-D2Rat157) using SHRSP as the recipient and WKY as the donor strain. The SP.WKYGla2k strain contains a 10-cM congenic interval, which is encompassed within the larger (64-cM) SP.WKYGla2a congenic region. Salt-loaded systolic blood pressure, measured by radiotelemetry, was significantly lower in the SP.WKYGla2a and SP.WKYGla2k strains compared with SHRSP. Salt sensitivity in SP.WKYGla2c* was not significantly different from SHRSP. Exclusion mapping identified a 6-Mbp region harboring genes responsible for salt-sensitive blood pressure regulation. Microarray expression profiling was carried out in whole homogenized kidneys from parental and SP.WKYGla2a strains. Examination of expression data within the minimal congenic interval identified the positional candidates Edg1 and Vcam1, demonstrating significantly elevated renal RNA expression levels in the SHRSP compared with WKY and SP.WKYGla2a congenic strains. These results were confirmed by quantitative real-time PCR. DNA sequencing identified SNPs in both Edg1 and Vcam1 between SHRSP and WKY rats. In conclusion, we have identified a suggestive minimal interval encompassing a 6-Mbp region on rat chromosome 2. This region contains several physiological candidate genes for salt-sensitive hypertension in the SHRSP, including Edg1 and Vcam1, which are differentially expressed and lie on common and functionally important pathways.
Key Words: hypertension • salt sensitivity • congenic • microarray expression profiling • candidate genes
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Introduction |
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Dietary sodium is an important contributor to essential hypertension with approximately one half of essential hypertensive patients demonstrating salt sensitivity.1,2 The potential importance of genetic factors in salt-sensitive hypertension has been demonstrated in both clinical studies3–5 and in the investigation of genetic rat models6–10; however, the underlying genetic determinants remain to be determined. Salt sensitivity enhances susceptibility to renal damage and is a major contributor to overall cardiovascular risk, particularly in aging populations.11,12 Enhanced sodium reabsorption, altered activity of neurohormonal systems, renal inflammation, and oxidative stress have all been implicated in this process.2,13–15
The stroke-prone spontaneously hypertensive rat (SHRSP) is a well-characterized experimental model for essential hypertension, demonstrating left ventricular hypertrophy, endothelial dysfunction, and salt sensitivity.16–18 The Wistar-Kyoto (WKY) rat, a strain of contrasting phenotype, demonstrates significantly less blood pressure response to salt.16 In a previous genome-wide scan of an F2 cross derived from the SHRSP and the WKY rat, we identified quantitative trait loci for baseline and salt-sensitive blood pressure on rat chromosome 2.16 These quantitative trait loci were subsequently confirmed with the use of reciprocal chromosome 2 congenic strains.18,19 Further studies, which combined congenic mapping and microarray analysis in the absence of salt loading, allowed identification of the functional candidate gene Gstm1,19,20 which is involved in the endogenous defense against oxidative stress. A similar combined strategy using salt-loaded congenic substrains will allow for identification of genetic factors responsible for salt sensitivity. The aim of this study was, therefore, to dissect the implicated locus within the SHRSP chromosome 2 congenic interval and to identify candidate genes for salt sensitivity based on microarray expression profiling and real-time PCR.
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Methods |
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Production of Congenic Strains
Inbred colonies of SHRSP and WKY rats have been maintained at the University of Glasgow since 1991, as described previously.16 From weaning, all of the rats were maintained on normal rat chow (rat and mouse No. 1 maintenance diet, Special Diet Services), and at 18 weeks of age, rats were given a salt challenge (1% NaCl in drinking water) for 3 weeks. Congenic strains SP.WKYGla2a (D2Rat13-D2Rat157) and SP.WKYGla2c* (D2Wox9-D2Mgh12) were generated using a marker-assisted "speed" congenic strategy18 where WKY (donor strain) segments were introgressed into the SHRSP (recipient strain) genetic background. The nomenclature of the strains consists of the first abbreviation belonging to the recipient strain and the second to the donor: Gla denotes that strains originate from the Glasgow colonies, and the number 2 refers to rat chromosome 2. Congenic substrains were generated by backcrossing male SP.WKYGla2a congenic rats with SHRSP females. Progeny generated from this backcross were heterozygous throughout the original congenic interval. Brother x sister mating was carried out to generate and fix substrains containing smaller congenic intervals, including SP.WKYGla2b (D2Rat13-D2Mit5), SP.WKYGla2e (D2Mit5-D2Rat133), SP.WKYGla2f (D2Wox15-D2Rat133), SP.WKYGla2g (D2Wox9-D2Rat231), SP.WKYGla2i (D2Rat132-D2Rat53), and SP.WKYGla2k (D2Mit21-D2Rat157; Figure 1).
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Strains SP.WKYGla2a, SP.WKYGla2b,18 and SP.WKYGla2c*19 have been published previously. All of the other substrains have been generated for the current project.
Hemodynamic Measurements
The Dataquest IV telemetry system (Data Sciences International) was used for the direct measurement of systolic blood pressure.16,18,21,22 Briefly, male rats were implanted at 12 weeks of age with 1-week recovery, 5 weeks of baseline measurements, followed by 3 weeks of 1% NaCl in the drinking water. Rats were killed at the end of the 21st week, and kidneys, spleen, and liver were snap frozen in liquid nitrogen and stored at –70°C for either RNA or DNA extraction. Genomic DNA was isolated from a 4-mm tip from the tail of congenic animals and genotyping as described previously.18 These studies were approved by the Home Office according to regulations regarding experiments with animals in the United Kingdom.
Gene Expression Profiling
Affymetrix GeneChip expression analysis was used to identify differentially expressed probe sets (representing a unique gene or expressed sequence tag sequence on the Affymetrix GeneChip) between male, 21-week, salt-loaded SHRSP, SP.WKYGla2a, and WKY rats. This design was used to maximize the potential for detection of genes that are differentially expressed and to map to the large congenic interval in the SP.WKYGla2a strain. Whole kidneys were homogenized and total RNA was extracted from 3 rats from each strain by using the maxi RNeasy kit according to the manufacturer’s protocol (Qiagen). Biotinylated, amplified target chromosomal RNA was prepared and hybridized to the Affymetrix Rat RAE230 A and B gene chips as described by Affymetrix.23 After hybridization, microarray chips were washed, stained, and scanned. Normalization was conducted using the Robust Multichip Average method24 implemented in the Affymetrix module in the Bioconductor microarray analysis software (http://www.bioconductor.org/), and differential expression was determined by Rank Products,25 where a 5% false discovery rate cutoff was used. For all of the probe sets with a false discovery rate <10%, a further 1000 permutations were carried out to obtain more precise P values and false discovery rates for those probe sets. The microarray data set has been submitted to ArrayExpress and can be accessed at http://www.ebi.ac.uk/ (Experimental Accession No. E-MIMR-541). Molecular interactions between genes were mapped to a common pathway using the Pathway Explorer function within Ingenuity Pathway Analysis software (Ingenuity Systems, www.ingenuity.com).
Quantitative Real-Time PCR
Renal total RNA was extracted from 21-week–old salt-loaded male rats using RNeasy kits (Qiagen), treated with DNase-Free RNase (Ambion), and accurately quantified using Ribogreen (Molecular Probes). Normalization was confirmed by performing real-time PCR (TaqMan, Applied Biosystems) of Actb (ß-actin; Promega) with comparable threshold cycles. TaqMan probes for Vcam1 (Rn01521370.m1-labeled FAM) or Edg1 (Rn02758712.s1-labeled FAM) were multiplexed with Actb (4352340E-labeled VIC). Expression of Vcam1 or Edg1 relative to Actb in each sample was derived using the comparative (
threshold change) method.
Sequencing
Renal RNA was used as a template for RT-PCR amplifying Edg1 and Vcam1 open reading frames from SHRSP and WKY strains. PCR primers were also designed to exon 1 and genomic DNA regions upstream of Edg1 and Vcam1 using the Brown Norway genome sequence as a template. PCR products were prepared with the Agencourt AMPure PCR Purification system (Agencourt BioScience) and sequenced with BigDye v3.1 fluorescent nucleotides (Applied Biosystems). Sequencing reactions were purified with the Agencourt CleanSEQ Sequencing Reaction Clean-Up system and run on the ABI 3730 using polymer 7 and the DNA sequence analyzed with SeqScape version 2.5 (Applied Biosystems).
Western Analysis of Endothelial Differentiation Gene Receptor 1 and Vascular Cell Adhesion Molecule 1 in Rat Kidney
Kidneys were removed rapidly from terminally anesthetized 21-week–old salt-loaded male SHRSP, SP.WKYGla2a, SP.WKYGla2k, and WKY rats. For endothelial differentiation gene receptor 1 (EDG1) western analysis, renal tissue was homogenized in protease inhibitor–containing buffer (50 mmol/L of HEPES, 150 mmol/L of NaCl, 1 mmol/L of dithiothreitol, and 0.5% v/v of Tween 20). For vascular cell adhesion molecule 1 (VCAM1) analysis, membrane- and cytoskeleton-enriched fractions were prepared with the cytoplasmic nuclear membrane compartment protein extraction kit as per the manufacturer’s protocol (AMS Biotechnology). Protein concentration was determined using a Bio-Rad bicinchoninic acid kit. Proteins were separated by 12% (EDG1) or 10% (VCAM1) polyacrylamide gel electrophoresis and electroblotted onto a Hybond-P membrane (Amersham). Membranes were incubated with the EDG1 SC-25489 (1:200) primary antibody (Santa Cruz Biotechnology) and VCAM1 SC-1504 (1:200) primary antibody (Santa Cruz Biotechnology) swine anti-rabbit 1:200 (PO399, Dakocytomation), and donkey anti-goat 1:1500 (ab7125, AbCam) horseradish peroxidase–conjugated secondary antibody, respectively. Protein loading was normalized with ß-actin 1:1000 (ab8226, AbCam) and incubated with horseradish peroxidase–conjugated rabbit anti-mouse secondary antibody 1:2000 (PO260, Dakocytomation). Protein bands were detected by chemiluminescence (ECL kit, Amersham) and visualized and quantified using a Bio-Rad Image Analyzer densitometry system (statistical analysis by ANOVA with Dunnett’s posthoc test).
Multianalyte Analysis of Rat Plasma Samples
Multianalyte analysis was performed on plasma samples from salt-loaded rats to assess circulating levels of proinflammatory cytokines and markers of renal injury. Plasma samples were obtained from male 21-week–old salt-loaded SHRSP, WKY, SP.WKYGla2a, and SP.WKYGla2k rats (n=4 per group). Samples were analyzed by Rules Based Medicine (Rules Based Medicine Inc) using a Luminex bead-based approach (http://www.rulesbasedmedicine.com/).
Statistical Analysis
All of the results are expressed as mean±SEM. Comparison of radiotelemetry data between congenic and parental strains was carried out by repeated-measures ANOVA, as described previously.21 F statistics and P values corresponding with the main effects for strain are reported. Comparison of data between congenic and parental strains for quantitative RT-PCR and Western densitometry analysis was carried out by 1-way ANOVA with Dunnett’s posthoc test for multiple comparisons.
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Results |
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A panel of congenic strains generated on an SHRSP background containing introgressed WKY segments of varying size, which provide overlapping coverage of the original SP.WKYGla2a congenic interval, is illustrated in Figure 1. Baseline and salt-loaded systolic blood pressure were not significantly different from that of parental SHRSP in SP.WKYGla2b, SP.WKYGla2f, SP.WKYGla2g, and SP.WKYGla2i strains (Figure 1). A reduction in systolic blood pressure in the SP.WKYGla2e congenic substrain reached borderline significance during baseline (F=4.82; P=0.041) and salt-loaded (F=4.08, P=0.059) periods. The SP.WKYGla2k substrain demonstrated significantly lower baseline (F=4.47; P=0.045) and salt-loaded systolic blood pressure (F=11.06; P=0.004). SP.WKYGla2k achieved an equivalent significant blood pressure reduction during salt loading to that of the SP.WKYGla2a congenic strain (Figure 2), indicating significantly reduced sensitivity to salt in the SP.WKYGla2k and SP.WKYGla2a strains. Averaged systolic blood pressure measurements for baseline and salt-loaded periods in SHRSP, SP.WKYGla2a, SP.WKYGla2c*, SP.WKYGla2k, and WKY strains are given in supplemental Table S1 (available online at http://hyper.ahajournals.org). These results suggest similar underlying genetic control and implicate the congenic interval, which is in common between the SP.WKYGla2a and SP.WKYGla2k strains. Conversely, the SP.WKYGla2c* strain retains its salt sensitivity, demonstrating no significant difference in salt-loaded systolic blood pressure to that of the SHRSP but demonstrating significantly elevated blood pressure in comparison with SP.WKYGla2a and SP.WKYGla2k strains (Figure 2 and Table S1).
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Increasing the density of polymorphic microsatellite markers within the lower region of rat chromosome 2 identified an 
6-Mb region (Figure 3), which was not in common between SP.WKYGla2c* and either SP.WKYGla2a or SP.WKYGla2k strains; ie, the lower boundary marker for SP.WKYGla2a and SP.WKYGla2k strains (D2Rat157) is 6-Mb distal to that of the lower boundary marker for SP.WKYGla2c* (D2Mgh12).
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Differential gene expression within the 6-Mb congenic interval was investigated by analysis of microarray expression profile data in whole homogenized kidneys from salt-loaded SHRSP, WKY, and SP.WKYGla2a strains. Of the 12 well-characterized National Center for Biotechnology Information RefSeq genes26 identified within this region (Figure 3) microarray analysis identified Edg1 and Vcam1 as having significantly lower expression levels in WKY and SP.WKYGla2a strains compared with SHRSP. A full list of renal microarray expression data for all of the probe sets identified within the 6-Mbp congenic interval are given in Table S2. The differential expression of Edg1 and Vcam1 in SHRSP, WKY, SP.WKYGla2a, and SP.WKYGla2k strains was confirmed by quantitative RT-PCR (Figure 4). In addition, several single nucleotide and insertion/deletion polymorphisms were identified in the regulatory regions of both Edg1 and Vcam1 genes between the SHRSPs and WKY rats (Table S3). SHRSP polymorphisms were confirmed in the spontaneously hypertensive rat (data not shown). Potential transcription factor–binding sites are given in Table S4. RT-PCR was not conducted in the remaining genes within this region because none demonstrated differential expression when assessed by microarray.
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To determine whether significantly increased expression of Edg1 and Vcam1 mRNA in SHRSPs resulted in significantly increased protein levels, western analysis was performed in kidneys from salt-loaded SHRSP, WKY, SP.WKYGla2a, and SP.WKYGla2k strains (Figure 5a and 5b, respectively). A single immunoreactive band was detectable for EDG1 and VCAM1 in each of the strains. Densitometry of the immunoblots identified a significant reduction in SHRSP EDG1 protein in comparison with SP.WKYGla2a and SP.WKYGla2k strains (Figure 5c). Densitometry of the immunoblots for VCAM1 identified significantly increased SHRSP VCAM1 protein levels in comparison with WKY, SP.WKYGla2a, and SP.WKYGla2k strains (Figure 5d). EDG1 and VCAM1 protein levels were not significantly different among SP.WKYGla2a, SP.WKYGla2k, and WKY strains.
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Levels of circulating markers of renal injury (cystatin C, osteopontin, neutrophil gelatinase-associated lipocalin, and clusterin) and proinflammatory mediators (soluble VCAM1, C-reactive protein, tumor necrosis factor-
, and fibrinogen) were elevated in plasma from SHRSP compared with WKY rats (Table).
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Discussion |
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The production of a panel of chromosome 2 congenic substrains has confirmed the existence of salt-sensitivity loci on rat chromosome 2 and allowed, by exclusion mapping, dissection of the implicated region to an
6-Mb interval. Moreover, a combined strategy of congenic substrain production and microarray expression profiling has identified 2 positional candidate genes (Edg1 and Vcam1) for salt-sensitive hypertension in the SHRSP. Both of these genes are linked through a number of common molecular networks (Figure 6).
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EDG1 (also known as sphingosine-1-phosphate 1) is a receptor for sphingosine-1-phosphate and belongs to the G protein–coupled receptor family. EDG1 has widespread distribution and is highly abundant on endothelial cells where it works in combination with EDG3 (sphingosine-1-phosphate 3) to regulate cell migration, differentiation, and endothelial barrier functions.27,28 Furthermore, studies using a knockout mouse model have shown Edg1 to be essential for vascular maturation.29 Altered Edg1 signaling has been implicated in the etiology of cardiovascular disorders, such as cardiac hypertrophy,30 inflammation, and atherosclerosis.31 Recent studies in rodent models of ischemia/reperfusion injury have shown that EDG1 agonists have a protective effect on renal function.32,33 This beneficial effect is associated with reduction of circulating lymphocytes, suppression of proinflammatory cytokines and adhesion molecules, and improved renal function with significantly reduced plasma creatinine levels.32 Results from the present study demonstrate significantly reduced EDG1 protein levels in kidneys from salt-loaded SHRSPs despite elevated mRNA expression. A lack of concordance between mRNA and protein levels is not unprecedented and possibly indicates abnormal posttranscriptional regulation or protein turnover in the SHRSP.34 This reduction in expression of EDG1 kidney protein may suggest reduced renoprotective ability in the SHRSP. Accordingly, we observed elevated levels of circulating markers of renal injury (cystatin C, osteopontin, clusterin, and neutrophil gelatinase-associated lipocalin) and proinflammatory mediators (C-reactive protein, fibrinogen, and tumor necrosis factor-) in plasma from SHRSP compared with WKY rats.
VCAM 1 exists as both membrane-bound and soluble forms. Membrane-bound VCAM1 allows the tethering of immune cells (eg, monocytes, lymphocytes, and leukocytes) to the endothelium and their transendothelial migration into the arterial intima.35 This early inflammatory response is central to the atherosclerotic process. The soluble form of VCAM1 serves as a monitor of membrane-bound VCAM1 expression on endothelial cells, smooth muscle cells, and macrophages, and increased levels reflect the progressive formation of atherosclerotic lesions.35,36
Rat models of hypertension, such as the SHRSP, do not normally develop atherosclerotic lesions, because the typical human lipoprotein profile is not displayed in rodents (ie, oxidized low-density lipoprotein levels are extremely low because plasma cholesterol is mainly transported in the form of high-density lipoprotein). Despite the lack of plaque development, increased expression of the soluble form of VCAM1 in the SHRSP may be indicative of an acute-phase response reflecting progressive, low-grade vessel wall or kidney inflammation. Indeed, previous studies in rats have indicated that a key mechanism for inducing salt sensitivity is the infiltration of inflammatory cells into the renal parenchyma.37
In this study, congenic substrains were used to narrow down the implicated congenic segment to aid the identification of positional candidate genes. Despite equivalent reductions in salt-loaded systolic blood pressure in both SP.WKYGla2a and SP.WKYGla2k strains compared with SHRSP, soluble VCAM1 levels were only significantly reduced in the SP.WKYGla2a strain. This result suggests that other factors outside of the SP.WKYGla2k region are important in the regulation of soluble form of VCAM1 levels and may indicate gene-gene interaction in the control of VCAM1 expression.
Activation of the Edg1 receptor and the regulation of Vcam1 expression are linked through a number of common intracellular signaling pathways38–41; therefore, investigation of the expression of other components of these pathways is warranted. Moreover, although this study implicates both Edg1 and Vcam1 in the development of salt-loaded hypertension, the present data cannot confirm whether their altered expression is a cause or consequence of hypertension itself, although some evidence of a causal role is implicated by the identification of SNPs and deletion/insertion mutations in the Edg1 and Vcam1 regulatory regions. However, these studies do not preclude the possibility of epistatic alleles or strain-specific differences that occur at the protein level that have not been detected by the microarray expression strategy.
Our previous congenic studies in the SHRSP identified Gstm1 as a positional and functional candidate gene for baseline hypertension on rat chromosome 2.19,20 The 2 candidate genes highlighted in this study, Vcam1 and Edg1, are located within the same chromosome but lie 8.7- and 8.3-Mb distal to Gstm1, respectively. Gstm1 is located outside of the SP.WKYGla2k 6-Mb implicated region and is, therefore, excluded as a contributing factor during salt-induced effects on blood pressure.
Perspectives
We have identified Edg1 and Vcam1 as positional and physiological candidates for salt sensitivity in the SHRSP and propose that altered signaling of the intracellular pathway linking these genes maybe implicated in the renal inflammation, oxidative stress, and endothelial dysfunction during salt loading. Based on these novel findings, the functionally important pathway between Edg1 and Vcam1 should now be prioritized for further study in rodent and human hypertension. This, in turn, will lead to the identification of new biomarkers for early diagnosis and novel therapeutic targets.
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Acknowledgments |
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Sources of Funding
This work was supported by the British Heart Foundation Chair (CH98001), programme grant funding (PG04/101 and RG/07/005/23633), the Wellcome Trust Cardiovascular Functional Genomics Initiative (066780/Z/01/Z), and the European Union Sixth Framework Programme Integrated Project (LSHG_CT 2005-019015 EURATools) awarded to A.F.D.
Disclosures
None.
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Footnotes |
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The first 2 authors contributed equally to this work.
Received May 25, 2007; first decision June 14, 2007; accepted September 18, 2007.
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