Transcriptome Sequencing to Detect the Potential Role of Long Noncoding RNAs in Salt-Sensitive Hypertensive Rats.

BACKGROUNDS: Long noncoding RNAs (lncRNAs) play an important role in various biological processes. However, their functions in salt-sensitive hypertension are largely unknown. In this study, the lncRNA-seq technique was employed to compare the expression profiles of lncRNAs and mRNAs in salt-sensitive hypertensive rats. METHODS: Blood pressure, serum sodium, and urinary creatinine were texted in salt-sensitive and salt-insensitive rats fed with different salt concentrations. High-throughput sequencing was used to detect the expression of lncRNAs and mRNA in the renal medulla of the two groups. RESULTS: Blood pressure and urinary sodium/creatinine of high-salt diets of the sensitive group were significantly higher than that in the control group. Serum sodium has no significant difference between the two groups in high-salt diets. NONRATG007131.2 and NONRATG012674.2 were the most different lncRNAs in the high salt-sensitive group. Correlation analysis reveals that Matn1, Serpinb12, Anxa8, and Hspa5 may play an important role in salt-sensitive hypertension. CONCLUSION: This study analyzed the difference in lncRNA and mRNA between salt-sensitive and salt-insensitive rats with different salt diets by high-throughput sequencing. Salt sensitivity and salt concentration were two key factors for the induction of hypertension. We found some potential genes that play an important role in salt-sensitive hypertension.

The novel organic mononitrate NDHP attenuates hypertension and endothelial dysfunction in hypertensive rats.

RATIONALE: Development and progression of cardiovascular diseases, including hypertension, are often associated with impaired nitric oxide synthase (NOS) function and nitric oxide (NO) deficiency. Current treatment strategies to restore NO bioavailability with organic nitrates are hampered by undesirable side effects and development of tolerance. In this study, we evaluated NO release capability and cardiovascular effects of the newly synthesized organic nitrate 1, 3-bis (hexyloxy) propan-2-yl nitrate (NDHP). METHODS: A combination of in vitro and in vivo approaches was utilized to assess acute effects of NDHP on NO release, vascular reactivity and blood pressure. The therapeutic value of chronic NDHP treatment was assessed in an experimental model of angiotensin II-induced hypertension in combination with NOS inhibition. RESULTS: NDHP mediates NO formation in both cell-free system and small resistance arteries, a process which is catalyzed by xanthine oxidoreductase. NDHP-induced vasorelaxation is endothelium independent and mediated by NO release and modulation of potassium channels. Reduction of blood pressure following acute intravenous infusion of NDHP was more pronounced in hypertensive rats (two-kidney-one-clip model) than in normotensive sham-operated rats. Toxicological tests did not reveal any harmful effects following treatment with high doses of NDHP. Finally, chronic treatment with NDHP significantly attenuated the development of hypertension and endothelial dysfunction in rats with chronic NOS inhibition and angiotensin II infusion. CONCLUSION: Acute treatment with the novel organic nitrate NDHP increases NO formation, which is associated with vasorelaxation and a significant reduction of blood pressure in hypertensive animals. Chronic NDHP treatment attenuates the progression of hypertension and endothelial dysfunction, suggesting a potential for therapeutic applications in cardiovascular disease.

Analysis of metabolites in plasma reveals distinct metabolic features between Dahl salt-sensitive rats and consomic SS.13(BN) rats.

Salt-sensitive hypertension is a major risk factor for cardiovascular disorders. Our previous proteomic study revealed substantial differences in several proteins between Dahl salt-sensitive (SS) rats and salt-insensitive consomic SS.13(BN) rats. Subsequent experiments indicated a role of fumarase insufficiency in the development of hypertension in SS rats. In the present study, a global metabolic profiling study was performed using gas chromatography/mass spectrometry (GC/MS) in plasma of SS rats (n=9) and SS.13(BN) rats (n=8) on 0.4% NaCl diet, designed to gain further insights into the relationship between alterations in cellular intermediary metabolism and predisposition to hypertension. Principal component analysis of the data sets revealed a clear clustering and separation of metabolic profiles between SS rats and SS.13(BN) rats. 23 differential metabolites were identified (P<0.05). Higher levels of five TCA cycle metabolites, fumarate, cis-aconitate, isocitrate, citrate and succinate, were observed in SS rats. Pyruvate, which connects TCA cycle and glycolysis, was also increased in SS rats. Moreover, lower activity levels of fumarase, aconitase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase were detected in the heart, liver or skeletal muscles of SS rats. The distinct metabolic features in SS and SS.13(BN) rats indicate abnormalities of TCA cycle in SS rats, which may play a role in predisposing SS rats to developing salt-sensitive hypertension.

FMRI and fcMRI phenotypes map the genomic effect of chromosome 13 in Brown Norway and Dahl salt-sensitive rats.

Genes have been implicated as major contributors to many biological traits and susceptibility to specific diseases. However, the mechanisms of genotype action on central nervous system function have been elusive. It has been previously observed that inbred Brown Norway (BN) rats exhibit a number of quantitative complex traits markedly different from those of inbred Dahl salt-sensitive (SS) rats. These strains have become so important to cardiovascular research that a novel chromosome substitution approach was used to create SS and BN strains that have a single chromosome replaced by the homologous chromosome of the other strain. The present study was conducted in an effort to evaluate whether fMRI neuroimaging measures could be employed as a phenotype of genetic influence on neural biology in SS, BN, and consomic SSBN13 rat strains. Electrical forepaw stimulation evoked robust differential BOLD-fMRI activation along the thalamocortical pathway among the three strains across different stimulus frequencies. Moreover, using the fMRI-guided seeds in thalamus and somatosensory cortex for the analysis of fcMRI, we were able to characterize the strain-specific difference in secondary somatosensory cortex, temporal association cortex, and the CA3 region. We were also able to define the genetic influences of Chr-13 on the projection and integration of sensory information in consomic SS-13(BN) strain. We provided objective imaging evidence supporting the hypothesis that rat strain-specific fMRI and fcMRI combined with consomic strategy can be a useful tool in identifying the complex genetic divergence that is related to neural circuits. These findings prove the concept of neuroimaging-based phenotypes as a novel approach to visualize and fine-map the genetic effects onto brain biology at a systems level.

SWI/SNF chromatin remodeling enzymes are associated with cardiac hypertrophy in a genetic rat model of hypertension.

Pathological cardiac hypertrophy is characterized by a sustained increase in cardiomyocyte size and re-activation of the fetal cardiac gene program. Previous studies implicated SWI/SNF chromatin remodeling enzymes as regulators of the fetal cardiac gene program in surgical models of cardiac hypertrophy. Although hypertension is a common risk factor for developing cardiac hypertrophy, there has not yet been any investigation into the role of SWI/SNF enzymes in cardiac hypertrophy using genetic models of hypertension. In this study, we tested the hypothesis that components of the SWI/SNF complex are activated and recruited to promoters that regulate the fetal cardiac gene program in hearts that become hypertrophic as a result of salt induced hypertension. Utilizing the Dahl salt-sensitive (S) rat model, we found that the protein levels of several SWI/SNF subunits required for heart development, Brg1, Baf180, and Baf60c, are elevated in hypertrophic hearts from S rats fed a high salt diet compared with normotensive hearts from Dahl salt-resistant (R) rats fed the same diet. Furthermore, we detected significantly higher levels of SWI/SNF subunit enrichment as well as evidence of more accessible chromatin structure on two fetal cardiac gene promoters in hearts from S rats compared with R rats. Our data implicate SWI/SNF chromatin remodeling enzymes as regulators of gene expression in cardiac hypertrophy resulting from salt induced hypertension. Thus we provide novel insights into the epigenetic mechanisms by which salt induced hypertension leads to cardiac hypertrophy.

Construction of two novel reciprocal conplastic rat strains and characterization of cardiac mitochondria.

Because of the lack of appropriate animal models, the potentially causal contributions of inherited mitochondrial genomic factors to complex traits are less well studied compared with inherited nuclear genomic factors. We previously detected variations between the mitochondrial DNA (mtDNA) of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). Specifically, multiple variations were detected in mitochondrial genes coding for subunits of proteins essential for electron transport, in mitochondrial reactive oxygen species production, and within the D-loop region. To evaluate the effects of these mtDNA variations in the absence of the corresponding nuclear genomic factors as confounding variables, novel reciprocal strains of S and SHR were constructed and characterized. When compared with that of the S rat, the heart tissue from the S.SHR(mt) conplastic strain wherein the mtDNA of the S rat was substituted with that of the SHR had a significant increase in mtDNA copy number and decrease in mitochondrial reactive oxygen species production. A corresponding increase in aerobic treadmill running capacity and a significant increase in survival that was not related to changes in blood pressure were observed in the S.SHR(mt) rats compared with the S rat. The reciprocal SHR.S(mt) rats did not differ from the SHR in any phenotype tested, suggesting lower penetrance of the S mtDNA on the nuclear genomic background of the SHR. These novel conplastic strains serve as invaluable tools to further dissect the relationship between heart function, aerobic fitness, cardiovascular disease progression, and mortality.

Genetic variants in Arhgef11 are associated with kidney injury in the Dahl salt-sensitive rat.

A previous genetic analysis comparing the Dahl salt-sensitive (S) rat with the spontaneously hypertensive rat identified a major locus on chromosome 2 that influences proteinuria in the S rat. In the present study, blood pressure, proteinuria, and renal hemodynamics were evaluated in congenic strains with small segments of the protective spontaneously hypertensive rat genome on the S background. Proteinuria and renal function were significantly improved in the congenic strains compared with the S. The causative locus interval was narrowed to <375 kb on the basis of congenic strains, haplotype data, comparative mapping, and concordance with human genetic studies. Sequencing of the coding region of genes in this region identified 36 single nucleotide polymorphisms (13 nonsynonymous and 23 synonymous). Gene expression profiling indicated that only a few genes exhibited differential expression. Arhgef11, Pear1, and Sh2d2 were identified as important candidate genes that may be linked to kidney injury in the S rat. In particular, Arhgef11 plays an important role in the activation of the Rho-ROCK signaling pathway. Inhibition of this pathway using fasudil resulted in a significant reduction of proteinuria in treated S rats (compared with untreated S). However, no difference was observed between treated or untreated spontaneously hypertensive rat or congenic strains. The homologous region in humans was found to be associated with estimated glomerular filtration rate in the Candidate Gene Association Resource population. In summary, these findings demonstrate that allelic variants in Arhgef11, acting through the Rho-ROCK pathway, could influence kidney injury in the S as well as provide insight into human kidney disease.

Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat.

The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.

Distinct QTLs cosegregate with worse hypertension and renal disease in ovariectomized F2[Dahl S × R]-intercross rats.

OBJECTIVES: Salt-sensitive hypertension is highly prevalent in postmenopausal women, with approximately 75% of postmenopausal women found to be hypertensive in the US. Insight from surgical menopause (ovariectomized) patients directly links the loss of endogenous estrogens to salt-sensitive hypertension in previously healthy, salt-resistant women. However, controversial benefit of hormone replacement therapy in postmenopausal women raises the hypothesis that the loss of endogenous estrogens alters genetic susceptibility determinants per se, resulting in hypertension mechanisms beyond correction by hormone replacement. METHODS: We studied ovariectomy-induced changes in hypertension phenotypes and performed a total genome scan for genetic determinants or quantitative trait loci (QTLs), which cosegregate with salt-sensitive hypertension and/or target organ complications in ovariectomized 6-month-old F2[Dahl S × R]-intercross female rats. We used SBP, glomerular injury score (GIS) and relative heart weight (RHW) as quantitative traits. We compared QTLs between ovariectomized and nonovariectomized F2[Dahl S × R]-intercross rats using identical phenotype and genotype characterization. RESULTS: Ovariectomy worsened hypertension and hypertensive nephrosclerosis but reduced RHW. Although some QTLs are common, hence ovarian hormone-independent, distinct BP-QTLs (on chromosomes 9, 13, 20 and X), RHW-QTLs (on chromosomes 1 and 3) and GIS-QTLs (on chromosomes 1 and 8) were detected in ovariectomized F2[Dahl S × R]-intercross female rats. CONCLUSION: Detection of worse hypertension phenotype and distinct QTLs in ovariectomized F2[Dahl S × R]-intercross female rats suggest that distinct genetic determinants underlie postmenopausal hypertension, which are activated, or de-repressed, upon the loss of estrogens.

Role of 20-HETE in the antihypertensive effect of transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats.

This study examined whether substitution of chromosome 5 containing the CYP4A genes from Brown Norway rat onto the Dahl S salt-sensitive (SS) genetic background upregulates the renal production of 20-HETE and attenuates the development of hypertension. The expression of CYP4A protein and the production of 20-HETE were significantly higher in the renal cortex and outer medulla of SS.5(BN) (chromosome 5-substituted Brown Norway rat) consomic rats fed either a low-salt (LS) or high-salt (HS) diet than that seen in SS rats. The increase in the renal production of 20-HETE in SS.5(BN) rats was associated with elevated expression of CYP4A2 mRNA. MAP measured by telemetry rose from 117 ± 1 to 183 ± 5 mmHg in SS rats fed a HS diet for 21 days, but only increased to 151 ± 5 mmHg in SS.5(BN) rats. The pressure-natriuretic and diuretic responses were twofold higher in SS.5(BN) rats compared with SS rats. Protein excretion rose to 354 ± 17 mg/day in SS rats fed a HS diet for 21 days compared with 205 ± 13 mg/day in the SS.5(BN) rats, and the degree of glomerular injury was reduced. Baseline glomerular capillary pressure (Pgc) was similar in SS.5(BN) rats (43 ± 1 mmHg) and Dahl S (44 ± 2 mmHg) rats. However, Pgc increased to 59 ± 3 mmHg in SS rats fed a HS diet for 7 days, while it remained unaltered in SS.5(BN) rats (43 ± 2 mmHg). Chronic administration of an inhibitor of the synthesis of 20-HETE (HET0016, 10 mg·kg(-1)·day(-1) iv) reversed the antihypertensive phenotype seen in the SS.5(BN) rats. These findings indicate that the transfer of chromosome 5 from the BN rat onto the SS genetic background increases the renal expression of CYP4A protein and the production of 20-HETE and that 20-HETE contributes to the antihypertensive and renoprotective effects seen in the SS.5(BN) consomic strain.

Transfer of the CYP4A region of chromosome 5 from Lewis to Dahl S rats attenuates renal injury.

This study examined the effect of transfer of overlapping regions of chromosome 5 that includes (4A(+)) or excludes (4A(-)) the cytochrome P-450 4A (CYP4A) genes from the Lewis rat on the renal production of 20-hydroxyeicosatetraenoic acid (20-HETE) and the development of hypertension-induced renal disease in congenic strains of Dahl salt-sensitive (Dahl S) rats. The production of 20-HETE was higher in the outer medulla of 4A(+) than in Dahl S or 4A(-) rats. Mean arterial pressure (MAP) rose to 190 +/- 7 and 185 +/- 3 mmHg in Dahl S and 4A(-) rats fed a high-salt (HS) diet for 21 days but only to 150 +/- 5 mmHg in the 4A(+) strain. Protein excretion increased to 423 +/- 40 and 481 +/- 37 mg/day in Dahl S and 4A(-) rats vs. 125 +/- 15 mg/day in the 4A(+) strain. Baseline glomerular capillary pressure (Pgc) was lower in 4A(+) rats (38 +/- 1 mmHg) than in Dahl S rats (42 +/- 1 mmHg). Pgc increased to 50 +/- 1 mmHg in Dahl S rats fed a HS diet, whereas it remained unaltered in 4A(+) rats (39 +/- 1 mmHg). Baseline glomerular permeability to albumin (P(alb)) was lower in 4A(+) rats (0.19 +/- 0.05) than in Dahl S or 4A(-) rats (0.39 +/- 0.02). P(alb) rose to approximately 0.61 +/- 0.03 in 4A(-) and Dahl S rats fed a HS diet for 7 days, but it remained unaltered in the 4A(+) rats. The expression of transforming growth factor-beta2 was higher in glomeruli of Dahl S rats than in 4A(+) rats fed either a low-salt (LS) or HS diet. Chronic administration of a 20-HETE synthesis inhibitor (HET0016; 10 mg.kg(-1).day(-1) sc) reversed the fall in MAP and renoprotection seen in 4A(+) rats. These results indicate that the introgression of the CYP4A genes from Lewis rats into the Dahl S rats increases the renal formation of 20-HETE and attenuates the development of hypertension and renal disease.

Blood pressure and proteinuria effects of multiple quantitative trait loci on rat chromosome 9 that differentiate the spontaneously hypertensive rat from the Dahl salt-sensitive rat.

A blood pressure (BP) quantitative trait locus (QTL) was previously located within 117 kb on rat chromosome 9 (RNO9) using hypertensive Dahl salt-sensitive and normotensive Dahl salt-resistant rats. An independent study between two hypertensive rat strains, the Dahl salt-sensitive rat and the spontaneously hypertensive rat (SHR), also detected a QTL encompassing this 117 kb region. Dahl salt-sensitive alleles in both of these studies were associated with increased BP. To map SHR alleles that decrease BP in the Dahl salt-sensitive rat, a panel of eight congenic strains introgressing SHR alleles onto the Dahl salt-sensitive genetic background were constructed and characterized. S.SHR(9)x3B, S.SHR(9)x3A and S.SHR(9)x2B, the congenic regions of which span a portion or all of the 1 logarithm of odds (LOD) interval identified by linkage analysis, did not significantly alter BP. However, S.SHR(9), S.SHR(9)x4A, S.SHR(9)x7A, S.SHR(9)x8A and S.SHR(9)x10A, the introgressed segments of which extend distal to the 1 LOD interval, significantly reduced BP. The shortest genomic segment, BP QTL1, to which this BP-lowering effect can be traced is the differential segment of S.SHR(9)x4A and S.SHR(9)x2B, to which an urinary protein excretion QTL also maps. However, the introgressed segment of S.SHR(9)x10A, located outside of this QTL1 region, represented a second BP QTL (BP QTL2) having no detectable effects on urinary protein excretion. In summary, the data suggest that there are multiple RNO9 alleles of the SHR that lower BP of the Dahl salt-sensitive rat with or without detectable effects on urinary protein excretion and that only one of these BP QTLs, QTL1, overlaps with the 117 kb BP QTL region identified using Dahl salt-sensitive and Dahl salt-resistant rats.

Genetic mapping of mammary tumor traits to rat chromosome 10 using a novel panel of consomic rats.

Identification of novel breast cancer susceptibility and resistance genes in genetically diverse human populations is challenging, and so inbred rats have been used to identify novel mammary cancer susceptibility quantitative trait loci (QTLs) with conventional mapping approaches. An alternative approach for QTL mapping is to use chromosome substitution (consomic) rat strains, which has the advantage of rapid generation of congenic from consomic animals. Using a novel rat strain pair, SS and BN, we identified rat mammary cancer QTLs in one of two consomic rat strains tested. Female rats of inbred parental (SS and BN) and two consomic (SS-10 BN and SS-12 BN) strains were treated with 7,12-dimethylbenz[a]anthracene orally. The phenotypes of tumor incidence, latency, and multiplicity were evaluated. SS rats were highly susceptible to mammary adenocarcinoma development, whereas BN rats were completely resistant. Statistical comparison of the phenotypes between the susceptible parental and the two consomic strains identified QTLs residing within chromosome 10 controlling mammary tumor latency and multiplicity. The study shows that SS-BN consomic rat strains can be used to map mammary tumor QTLs. This novel approach should accelerate positional cloning of mammary cancer susceptibility and resistant genes in the rat and the identification of homologous genes in humans.

Distinct genomic replacements from Lewis correct diastolic dysfunction, attenuate hypertension, and reduce left ventricular hypertrophy in Dahl salt-sensitive rats.

BACKGROUND: Hypertension and diastolic heart failure are two common cardiovascular diseases that inflict heavy morbidity and mortality, yet relatively little is understood about their pathophysiology. The identification of quantitative trait loci for blood pressure is important in unveiling the causes of polygenic hypertension. Although Dahl salt-sensitive strain is also an excellent model for the study of diastolic heart failure, virtually nothing is known about the quantitative trait loci determining diastolic heart failure. Diastolic dysfunction often represents the onset of diastolic heart failure. METHODS: We first characterized the cardiac phenotype of Dahl salt-sensitive strain and normotensive Lewis control rats by echocardiography to ascertain diastolic function. We then analyzed corresponding features of four newly developed and two existing congenic strains, each of which carries a specific chromosome substitution of Dahl salt-sensitive strain by its Lewis homologue and each lowering blood pressure. RESULTS: Dahl salt-sensitive strain displayed diastolic dysfunction that was rectified in two of six congenic strains, designated as positive congenic strains, which represent the first rodent models exhibiting functional normalization of diastolic dysfunction caused by naturally occurring genetic variants. The two positive congenic strains also showed a reduction in left ventricular mass. In contrast, four of six congenic strains did not change diastolic function despite their blood pressure-lowering effects. CONCLUSION: Genes present in the replaced chromosome segments of the two positive congenic strains are not commonly known to affect blood pressure, diastolic function or left ventricular mass. Consequently, novel prognostic, diagnostic and therapeutic strategies for hypertensive diastolic heart failure likely emerge from this work.

Important genetic checkpoints for insulin resistance in salt-sensitive (S) Dahl rats.

Despite the marked advances in research on insulin resistance (IR) in humans and animal models of insulin resistance, the mechanisms underlying high salt-induced insulin resistance remain unclear. Insulin resistance is a multifactorial disease with both genetic and environmental factors (such as high salt) involved in its pathogenesis. High salt triggers insulin resistance in genetically susceptible patients and animal models of insulin resistance. One of the mechanisms by which high salt might precipitate insulin resistance is through its ability to enhance an oxidative stress-induced inflammatory response that disrupts the insulin signaling pathway. The aim of this hypothesis is to discuss two complementary approaches to find out how high salt might interact with genetic defects along the insulin signaling and inflammatory pathways to predispose to insulin resistance in a genetically susceptible model of insulin resistance. The first approach will consist of examining variations in genes involved in the insulin signaling pathway in the Dahl S rat (an animal model of insulin resistance and salt-sensitivity) and the Dahl R rat (an animal model of insulin sensitivity and salt-resistance), and the putative cellular mechanisms responsible for the development of insulin resistance. The second approach will consist of studying the over-expressed genes along the inflammatory pathway whose respective activation might be predictive of high salt-induced insulin resistance in Dahl S rats. Variations in genes encoding the insulin receptor substrates -1 and/or -2 (IRS-1, -2) and/or genes encoding the glucose transporter (GLUTs) proteins have been found in patients with insulin resistance. To better understand the combined contribution of excessive salt and genetic defects to the etiology of the disease, it is essential to investigate the following question:Question 1: Do variations in genes encoding the IRS -1 and -2 and/or genes encoding the GLUTs proteins predict high salt-induced insulin resistance in Dahl S rats?A significant amount of evidence suggested that salt-induced oxidative stress might predict an inflammatory response that upregulates mediators of inflammation such as the nuclear factor- kappa B (NF-kappa B), the tumor necrosis factor-alpha (TNF-alpha) and the c-Jun Terminal Kinase (JNK). These inflammatory mediators disrupt the insulin signaling pathway and predispose to insulin resistance. Therefore, the following question will be thoroughly investigated:Question 2: Do variations in genes encoding the NF-kappa B, the TNF-alpha and the JNK, independently or in synergy, predict an enhanced inflammatory response and subsequent insulin resistance in Dahl S rats in excessive salt environment?Finally, to better understand the combined role of these variations on glucose metabolism, the following question will be addressed:Question 3: What are the functional consequences of gene variations on the rate of glucose delivery, the rate of glucose transport and the rate of glucose phosphorylation in Dahl S rats?The general hypothesis is that "high-salt diet in combination with defects in candidate genes along the insulin signaling and inflammatory pathways predicts susceptibility to high salt-induced insulin resistance in Dahl S rats".

Genetic and dietary salt contributors to insulin resistance in Dahl salt-sensitive (S) rats.

Insulin resistance has been extensively investigated during the past decade because of its proposed role in initiating a cluster of cardiovascular risk factors including hypertension. Insulin resistance is an inherited genetic trait that precedes hypertension in Dahl salt-sensitive (S) rats, and is not present in Dahl salt-resistant (R) rats. Owing to the co-existence of insulin resistance and salt sensitivity of blood pressure in Dahl S, but not R rats, Dahl S rats are used to elucidate the role of dietary salt as a potential link in exacerbating both phenotypes (insulin resistance and salt sensitivity). In light of available data, examining the impact of dietary salt on insulin resistance in Dahl S rats in terms of salt concentration and duration of exposure helps answer the following question: What percentage of dietary salt and for what duration of exposure would we expect an enhanced insulin resistance in Dahl S rats? This commentary gathers all available research done on insulin resistance in Dahl S rats in an attempt to unravel dietary salt contribution to insulin resistance in Dahl S rats.

Submegabase resolution of epistatically interacting quantitative trait loci for blood pressure applicable for essential hypertension.

OBJECTIVE: Although genetic mapping of quantitative trait loci for blood pressure to large chromosome segments is readily achievable, their final identification confronts formidable hurdles. Restriction of the genes lodging in one quantitative trait locus interval to experimental limitation can facilitate their positional cloning. We previously delineated several quantitative trait loci for blood pressure on chromosome 10 of Dahl salt-sensitive rats, but their chromosome delimitations were either large or not definitive. METHODS: In this study, we systematically and comprehensively constructed congenic strains with submegabase (Mb) genome resolution and analyzed their blood pressure by telemetry. RESULTS: Three quantitative trait loci have been conclusively delimited by three congenic strains, each independently lowering the blood pressure. Their intervals are demarcated by genomic regions between 350 and 910 kilobases (kb) in size. Two of the three quantitative trait loci share an epistatic relationship and are separated from one another by less than 170 kb. Two additional quantitative trait loci for blood pressure were also tentatively delineated and their intervals range from 520 kb to 1.75 Mb. Possible genes dwelling in each quantitative trait locus-interval number between 11 and 17. None of these genes is known to exert a functional impact on blood pressure. Work is underway to find candidate genes with mutations that could be responsible for the blood pressure effect. CONCLUSION: Novel diagnostic, prognostic, preventive and/or therapeutic targets for essential hypertension and hypertension-associated diseases are likely to emerge from the identification of these quantitative trait loci. Potential applications of these quantitative trait loci to humans are suggested from the positive results from several association studies, demonstrating the existence of quantitative trait loci in the broad homologous regions.

The Atp1a1 gene from inbred Dahl salt sensitive rats does not contain the A1079T missense transversion.

The existence of the A1079T transversion in the alpha1 isoform of the Na(+), K(+)-ATPase (Atp1a1) gene in Dahl salt-sensitive rat (SS/Jr) strain, discovered by Herrera and Ruiz-Opazo and proposed to underlay hypertension sensitivity, represents one of the most controversial topics in hypertension research. As our research group did not have any previous connection to any party in this dispute nor to hypertension-related research, we were asked (J Hypertens. 2006;24:2312-2313) to definitively adjudge the existence of the A1079T transversion. Hence, different state-of-the art SNP detection technologies that depend on a variety of mechanisms and enzymes to detect the transversion in genomic DNA as well as cDNA derived from different tissues were used. Although it was possible to readily detect other silent polymorphisms between SS and SR strains in the Atp1a1 gene by all methods used, no evidence for the existence of the A1079T transversion in SS/Jr rats was found.

Multiple blood pressure loci on rat chromosome 13 attenuate development of hypertension in the Dahl S hypertensive rat.

Previous studies have indicated that substitution of chromosome 13 of the salt-resistant Brown Norway BN/SsNHsdMcwi (BN) rat into the genomic background of the Dahl salt-sensitive SS/JrHsdMcwi (SS) rat attenuates the development of salt-sensitive hypertension and renal damage. To identify the regions within chromosome 13 that attenuate the development of hypertension during a high-salt diet in the SS rat, we phenotyped a series of overlapping congenic lines covering chromosome 13, generated from an intercross between the consomic SS-13(BN) rat and the SS rat. Blood pressure was determined in chronically catheterized rats after 2 wk of high-salt diet (8% NaCl) together with microalbuminuria as an index of renal damage. Four discrete regions were identified, ranging in size from 4.5 to 16 Mbp, each of which independently provided significant protection from hypertension during high-salt diet, reducing blood pressure by 20-29 mmHg. Protection was more robust in female than male rats in some of the congenic strains, suggesting a sex interaction with some of the genes determining blood pressure during high-salt diet. Among the 23 congenic strains, several regions overlapped. When three of the "protective" regions were combined onto one broad congenic strain, no summation effect was seen, obtaining the same decrease in blood pressure as with each one independently. We conclude from these studies that there are four regions within chromosome 13 containing genes that interact epistatically and influence arterial pressure.