[Show abstract][Hide abstract] ABSTRACT: Background – Genome wide association studies (GWAS) are powerful tools for nominating pathogenic variants, but offer little insight as to how candidate genes impact disease outcome. Such is the case for SH2B adaptor protein 3 (SH2B3), which is a negative regulator of multiple cytokine signaling pathways and is associated with increased risk of myocardial infarction (MI), but its role in post-MI inflammation and fibrosis is completely unknown.
Methods and Results ¬– Using an experimental model of MI (left anterior descending artery [LAD] occlusion) in wild-type (WT) and Sh2b3 knockout rats (Sh2b3em2Mcwi), we assessed the role of Sh2b3 in post-MI fibrosis, leukocyte infiltration, angiogenesis, left ventricle (LV) contractility, and inflammatory gene expression. Compared with WT, Sh2b3em2Mcwi rats had significantly increased fibrosis (2.2-fold; P<0.05) and elevated leukocyte infiltration (>2-fold; P<0.001), which coincided with decreased LV fractional shortening (FS) (-∆11%; P<0.05) at 7 days post-LAD occlusion/reperfusion injury. Despite an increased angiogenic potential in Sh2b3em2Mcwi rats (1.7-fold; P<0.05), we observed no significant differences in LV capillary density between WT and Sh2b3em2Mcwi rats. In total, 12 genes were significantly elevated in the post-LAD occluded/reperfused hearts of Sh2b3em2Mcwi rats relative to WT, of which three (NLRP12, CCR2, and IFNγ) were significantly elevated in the LV of heart failure (HF) patients carrying the MI-associated rs3184504 [T] SH2B3 risk allele.
Conclusions – These data demonstrate for the first time that SH2B3 is a crucial mediator of post-MI inflammation and fibrosis.
[Show abstract][Hide abstract] ABSTRACT: The classic renin-angiotensin system is partly responsible for controlling aldosterone secretion from the adrenal cortex via the peptide angiotensin II (AngII). In addition, there is a local adrenocortical renin-angiotensin system that may be involved in the control of aldosterone synthesis in the zona glomerulosa (ZG). In order to characterize the long-term control of adrenal steroidogenesis, we utilized adrenal glands from renin knockout (KO) rats and compared steroidogenesis in vitro and steroidogenic enzyme expression to wild-type (WT) controls (Dahl S rat). Adrenal capsules (ZG; aldosterone production) and subcapsules (zona reticularis/fasciculata [ZFR]; corticosterone production) were separately dispersed and studied in vitro. Plasma renin activity and angiotensin II concentrations were extremely low in the KO rats. Basal and cAMP-stimulated aldosterone production was significantly reduced in renin KO ZG cells whereas corticosterone production was not different between WT and KO ZFR cells. As expected, adrenal renin mRNA expression was lower in the renin KO compared to the WT rat. Real-time PCR and immunohistochemical analysis showed a significant decrease in P450aldo (Cyp11b2) mRNA and protein expression in the ZG from the renin KO rat. The reduction in aldosterone synthesis in the ZG of the renin KO adrenal seems to be accounted for by a specific decrease in P450aldo, and may be due to the absence of chronic stimulation of the ZG by circulating AngII or to a reduction in locally-released AngII within the adrenal gland.
[Show abstract][Hide abstract] ABSTRACT: The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that impact only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the non-malignant microenvironment. Using CXM, we defined genetic variant(s) on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3IL2Rγ consomic model compared to the SSIL2Rγ parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3IL2Rγ background. Through comparative mapping and whole genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment which underlie differences in breast cancer risk.
[Show abstract][Hide abstract] ABSTRACT: PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.
Proceedings of the National Academy of Sciences 08/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The goal of the present study was to narrow a region of chromosome 13 to only several genes and then apply unbiased statistical approaches to identify molecular networks and biological pathways relevant to blood pressure salt-sensitivity in Dahl salt-sensitive rats. The analysis of 13 overlapping subcongenic strains identified a 1.37 Mbp region on chromosome 13 that influenced the mean arterial blood pressure by at least 25 mmHg in SS rats fed a high salt diet. DNA sequencing and analysis filled genomic gaps and provided identification of five genes in this region, Rfwd2, Fam5b, Astn1, Pappa2, and Tnr. A cross-platform normalization of transcriptome data sets obtained from our previously published Affymetrix GeneChip dataset and newly acquired RNA-seq data from renal outer medullary tissue provided 90 observations for each gene. Two Bayesian methods were used to analyze the data: 1) A linear model analysis to assess 243 biological pathways for their likelihood to discriminate blood pressure levels across experimental groups; 2) A Bayesian graphical modeling of pathways to discover genes with potential relationships to the candidate genes in this region. As none of these five genes are known to be involved in hypertension, this unbiased approach has provided useful clues to be experimentally explored. Of these five genes, Rfwd2, the gene most strongly expressed in the renal outer medulla, was notably associated with pathways that can affect blood pressure via renal transcellular Na(+) and K(+) electrochemical gradients and tubular Na+ transport, mitochondrial TCA cycle and cell energetics, and circadian rhythms.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locus, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. We used gene-targeting in a disease-susceptible rat model of genetic hypertension to test all six genes at the AGTRAP-PLOD1 locus (AGTRAP, MTHFR, CLCN6, NPPA, NPPB, and PLOD1) in blood pressure (BP) and renal phenotypes. This revealed that the majority of genes at this locus (5 out of 6) can impact hypertension by modifying BP and renal phenotypes. Mutations of NPPA, PLOD1, and MTHFR increased disease susceptibility, whereas AGTRAP and CLCN6 mutations decreased hypertension risk. Re-analysis of the human AGTRAP-PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were not only possible, but rather were highly plausible. Combined, these data demonstrate for the first time that multiple modifiers of hypertension can cosegregate at a single GWAS locus.
[Show abstract][Hide abstract] ABSTRACT: A 3.7-Mb region of rat chromosome 13 (45.2-49.0 Mb) affects blood pressure (BP) in females only, indicating the presence of sex-specific BP loci in close proximity to the Renin locus. In the present study, we used a series of Dahl salt-sensitive/Mcwi-13 Brown Norway congenic rat strains to further resolve BP loci within this region. We identified 3 BP loci affecting female rats only, of which the 2 smaller loci (line9BP3 and line9BP4) were functionally characterized by sequence and expression analysis. Compared with SS (SS/HsdMcwiCrl), the presence of a 591-kb region of BN (BN/NHsdMcwi) chromosome 13 (line9BP3) significantly lowered BP by 21 mm Hg on an 8% NaCl diet (153±7 versus 174±5 mm Hg; P<0.001). Unexpectedly, the addition of 23 kb of Brown Norway chromosome 13 (line9BP4) completely erased the female-specific BP protection on 8% NaCl diet, suggesting that BN hypertensive allele(s) reside in this region. The congenic interval of the protective line 9F strain contains 3 genes (Optc, Prelp, and Fmod), and the hypertensive line 9E contains 1 additional gene (Btg2). Sequence analysis of the 2 BP loci revealed a total of 282 intergenic variants, with no coding variants. Analysis of gene expression by quantitative real-time polymerase chain reaction revealed strain- and sex-specific differences in Prelp, Fmod, and Btg2 expression, implicating these as novel candidate genes for female-specific hypertension.
[Show abstract][Hide abstract] ABSTRACT: Many lines of evidence demonstrate that genetic variability contributes to chronic kidney disease susceptibility in humans as well as rodent models. Little progress has been made in discovering causal kidney disease genes in humans mainly due to genetic complexity. Here, we use a minimal congenic mapping strategy in the FHH (Fawn Hooded Hypertensive) rat to identify Sorcs1 as a novel renal disease candidate gene. We investigated the hypothesis that genetic variation in Sorcs1 influences renal disease susceptibility in both rat and human. Sorcs1 is expressed in the kidney, and knocking out this gene in a rat strain with a sensitized genome background produced increased proteinuria. In vitro knockdown of Sorcs1 in proximal tubule cells impaired protein trafficking, suggesting a mechanism for the observed proteinuria in the FHH rat. Since Sorcs1 influences renal function in the rat, we went on to test this gene in humans. We identified associations between single nucleotide polymorphisms (SNPs) in SORCS1 and renal function in large cohorts of European and African ancestry. The experimental data from the rat combined with association results from different ethnic groups indicates a role for SORCS1 in maintaining proper renal function.
[Show abstract][Hide abstract] ABSTRACT: Plekha7 (Plekstrin homology domain containing family A member 7) has been identified by numerous genetic studies as a candidate gene for human hypertension. We disrupted Plekha7 in the SS rat by Zinc-Finger Nuclease (ZFN) mutagenesis in order to investigate its role in blood pressure control. Four weeks on a high salt diet (8% NaCl) produced no changes in blood pressure between mutant and wild-type (WT) littermates, although mutants demonstrated improved renal phenotypes (reduced proteinuria, protein casting, glomerulosclerosis, and fibrosis). We hypothesized that these differences were caused by vascular changes in the Plekha7 mutants. Resistance arteries from these mutants were less responsive to dilators acetylcholine (logEC50 of –6.01±0.17 vs. – 6.66±0.12, p≤0.05) and nitroprusside (logEC50 of –7.19±0.14 vs. – 7.66±0.13, p≤0.05) but showed no difference in contractile response to phenylephrine (PE) or endothelin-1. In Ca2+-free buffer, vessels contracted more to 10 μM PE (32.44±2.08% vs. 15.58±2.89%, p≤0.05), indicating an elevation in intracellular Ca2+ content. In isolated glomeruli, there was an amplified intracellular Ca2+ response to high extracellular Ca2+. These data indicate that despite a lack of evidence for controlling blood pressure in the SS rat, Plekha7 plays an integral role in vascular function through regulation of intracellular Ca2+.
[Show abstract][Hide abstract] ABSTRACT: Renin was the first blood pressure (BP) quantitative trait locus mapped by linkage analysis in the rat. Subsequent BP linkage and congenic studies capturing different portions of the renin region have returned conflicting results, suggesting that multiple interdependent BP loci may be residing in the chromosome 13 BP quantitative trait locus that includes Renin. We used SS-13BN congenic strains to map 2 BP loci in the Renin region (chr13: 45.2-49.0 Mb). We identified a 1.1-Mb protective Brown Norway region around Renin (chr13: 46.1-47.2 Mb) that significantly decreased BP by 32 mm Hg. The Renin protective BP locus was offset by an adjacent hypertensive locus (chr13: 47.2-49.0 Mb) that significantly increased BP by 29 mm Hg. Sequence analysis of the protective and hypertensive BP loci revealed 1433 and 2063 variants between Dahl salt-sensitive/Mcwi and Brown Norway rats, respectively. To further reduce the list of candidate variants, we regenotyped an overlapping SS-13SR congenic strain (S/renrr) with a previously reported BP phenotype. Sequence comparison among Dahl salt-sensitive, Dahl R, and Brown Norway reduced the number of candidate variants in the 2 BP loci by 42% for further study. Combined with previous studies, these data suggest that at least 4 BP loci reside within the 30-cM chromosome 13 BP quantitative trait locus that includes Renin.
[Show abstract][Hide abstract] ABSTRACT: We previously reported that the fawn-hooded hypertensive (FHH) rat is a natural Rab38 knockout, supported by a congenic animal (FHH.BN-Rab38) having less proteinuria than FHH animals. Because these congenic animals contain Brown Norway (BN) alleles for five other named genes; however, a causal role for Rab38 in the FHH phenotype remains uncertain. Here, we used transgenic and knockout models to validate Rab38 and to exclude other genes within the 1.5 Mb congenic region from involvement in causing the FHH phenotype. Transgenic rats homozygous for the wild-type Rab38 BN allele on the FHH background exhibited phenotypic rescue, having 43% lower proteinuria and 75% lower albuminuria than nontransgenic FHH littermates. Conversely, knockout of the Rab38 gene on the FHH.BN-Rab38 congenic line recapitulated a proteinuric phenotype indistinguishable from the FHH strain. In addition, in cultured proximal tubule LLC-PK1 cells, knockdown of Rab38 mRNA significantly decreased endocytosis of colloidal gold-coupled albumin, supporting the hypothesis that Rab38 modulates proteinuria through effects on tubular re-uptake and not by altering glomerular permeability. Taken together, these findings validate Rab38 as a gene having a causal role in determining the phenotype of the FHH rat, which models hypertension-associated renal disease. Furthermore, our data suggest that Rab38 affects urinary protein excretion via effects in the proximal tubule.
Journal of the American Society of Nephrology 01/2013; · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.-Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bősze, Z., Rülicke, T., Izsvák, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.
[Show abstract][Hide abstract] ABSTRACT: Studies of transcriptome profiles have provided new insights into mechanisms underlying the development of hypertension. Cell type heterogeneity in tissue samples, however, has been a significant hindrance in these studies. We performed a transcriptome analysis in medullary thick ascending limbs of the loop of Henle isolated from Dahl salt-sensitive rats. Genes differentially expressed between Dahl salt-sensitive rats and salt-insensitive consomic SS.13(bn) rats on either 0.4% or 7 days of 8.0% NaCl diet (n=4) were highly enriched for genes located on chromosome 13, the chromosome substituted in the SS.13(bn) rat. A pathway involving cell proliferation and cell cycle regulation was identified as one of the most highly ranked pathways based on differentially expressed genes and by a Bayesian model analysis. Immunofluorescent analysis indicated that just 1 week of a high-salt diet resulted in a severalfold increase in proliferative medullary thick ascending limb cells in both rat strains, and that Dahl salt-sensitive rats exhibited a significantly greater proportion of medullary thick ascending limb cells in a proliferative state than in SS.13(bn) rats (15.0±1.4% versus 10.1±0.6%; n=7-9; P<0.05). The total number of cells per medullary thick ascending limb section analyzed was not different between the 2 strains. The study revealed alterations in regulatory pathways in Dahl salt-sensitive rats in tissues highly enriched for a single cell type, leading to the unexpected finding of a greater increase in the number of proliferative medullary thick ascending limb cells in Dahl salt-sensitive rats on a high-salt diet.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have identified multiple blood pressure and renal disease quantitative trait loci located on rat chromosome 12. In the present study, we narrowed blood pressure loci using a series of overlapping Dahl salt-sensitive/Mcwi (SS)-12 Brown Norway (BN) congenic lines. We found that transferring 6.1 Mb of SS chromosome 12 (13.4-19.5 Mb) onto the consomic SS-12(BN) background significantly elevated blood pressure on 1% NaCl (146 ± 6 versus 127 ± 1 mm Hg; P<0.001) and 8% NaCl diets (178 ± 7 versus 144 ± 2 mm Hg; P<0.001). Compared with the SS-12(BN) consomic, these animals also had significantly elevated albumin (218 ± 31 versus 104 ± 8 mg/d; P<0.001) and protein excretion (347 ± 41 versus 195 ± 12 mg/d; P<0.001) on a 1% NaCl diet. Elevated blood pressure, albuminuria, and proteinuria coincided with greater renal and cardiac damage, demonstrating that SS allele(s) within the 6.1 Mb congenic interval are associated with strong cardiovascular disease phenotypes. Sequence analysis of the 6.1 Mb congenic region revealed 12 673 single nucleotide polymorphisms between SS and BN rats. Of these polymorphisms, 293 lie within coding regions, and 18 resulted in nonsynonymous changes in conserved genes, of which 5 were predicted to be potentially damaging to protein function. Syntenic regions in human chromosome 7 have also been identified in multiple linkage and association studies of cardiovascular disease, suggesting that genetic variants underlying cardiovascular phenotypes in this congenic strain can likely be translated to a better understanding of human hypertension.
[Show abstract][Hide abstract] ABSTRACT: Human data and animal models of autosomal recessive polycystic kidney disease (ARPKD) suggest that genetic factors modulate the onset and severity of the disease. We report here for the first time that ARPKD susceptibility is attenuated by introgressing the mutated Pkhd1 disease allele from the polycystic kidney (PCK) rat onto the FHH (Fawn-Hooded Hypertensive) genetic background. Compared with PCK, the FHH.Pkhd1 strain had significantly decreased renal cyst formation that coincided with a threefold reduction in mean kidney weights. Further analysis revealed that the FHH. Pkhd1 is protected from increased blood pressure as well as elevated plasma creatinine and blood urea nitrogen levels. On the other hand, liver weight and biliary cystogenesis revealed no differences between PCK and FHH.Pkdh1, indicating that genes within the FHH genetic background prevent the development of renal, but not hepatic, manifestations of ARPKD. Microarray expression analysis of kidneys from 30-day-old PCK rats revealed increased expression of genes previously identified in PKD renal expression profiles, such as inflammatory response, extracellular matrix synthesis, and cell proliferation genes among others, whereas the FHH.Pkhd1 did not show activation of these common markers of disease. This newly developed strain can serve as a tool to map modifier genes for renal disease in ARPKD and provides further insight into disease variability and pathophysiology.
[Show abstract][Hide abstract] ABSTRACT: The combined transfer of two renal function quantitative trait loci (QTLs), Rf-1 (rat chromosome 1) and Rf-4 (rat chromosome 14), from the Fawn-hooded hypertensive rat onto the August Copenhagen Irish genetic background significantly increases proteinuria and demonstrates an interaction between these QTLs. Because the original Rf-4 congenic region is 61.9 Mbp, it is necessary to reduce this interval to feasibly search for variants responsible for renal susceptibility in this region. Here, we generated a minimal congenic line (Rf-1a+4_a) to identify a 4.1-Mb region of the Rf-4 QTL that significantly contributes to the severity of proteinuria in the Fawn-hooded hypertensive rat. Rf-1a+4_a animals have an increased glomerular permeability to albumin without significant changes in BP, indicating that at least one genetic element in this refined region directly affects renal function. Sequence analysis revealed no variants predicted to damage protein function, implying that regulatory elements are responsible for the Rf-4 phenotype. Multiple human studies, including recent genome-wide association studies, link the homologous human region with susceptibility to renal disease, suggesting that this congenic line is an important model for studying pathways that contribute to the progression of kidney disease.
Journal of the American Society of Nephrology 02/2012; 23(5):825-33. · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Human genome wide association (GWAS) and gene-centric studies show association of chromosomal regions or single nucleotide polymorphisms (SNPs) with hypertension. Unfortunately, progress in finding causal genes and SNPs has been limited. Alternative methods are required for dissection of complex diseases like hypertension. Various studies have shown that animal models of cardiovascular disease, for which rats are extensively characterized, can serve to find potential therapeutic targets. Using new high throughput sequencing technologies we analyzed rat genomic regions homologous to reproducible regions identified by human studies on the genomic and RNA level. Methods: The genomic DNA of hypertensive rat strains and the associated normotensive strains was sequenced on an Illumina HiSeq 2000. To enhance alignment of the whole genome data, large insert libraries were also sequenced as well as regional sequencing using a Roche 454 GS and PacBio RS. Transcriptome data were generated by sequencing the mRNA from heart, kidney, liver and adrenal gland. Results: Examination of all hypertensive crosses for three QTL regions found no evidence of a conserved haplotype, suggesting the involvement of multiple genes within a common QTL. Transcriptome analysis revealed strain dependent isoforms as well as clustering of transcript by tissue across the genome. Conclusions: The lack of conserved haplotypes among hypertensive strains indicates complex, strain dependent gene interactions contributing to the hypertension. The genetic complexity between rat strains mimics human population differences, with the benefit of an inbred, environment controlled animal model.
Journal of Hypertension 01/2012; 30:e40. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Despite significant heritability and extensive effort to identify genes contributing to human hypertension, limited progress has been made to find causal genes partially as a result of multiple variants each having a small effect. Therefore, there is a need to pursue alternative strategies for identifying genes contributing to hypertension. With the advent of new technologies to manipulate the rat genome, systematic modification of a sequence variant or variants in chromosomal regions nominated by human studies can be accomplished. Methods: To elucidate the role of genes identified in human blood pressure (BP) regulation GWAS, zinc-finger nuclease (ZFN) technology was used to create mutant strains for over 70 genes in different inbred rat strains. In each strain BP and proteinuria were measured following exposure to 4% salt diet. Sequencing of genomic DNA was performed on strains utilized in generating ZFN mutants and the corresponding normotensive strains. Results: Analysis of mutant strains that resulted in changes in BP revealed involvement of different genes in the ERK/MAPK Signalling pathway. Sequence analysis of the regions of interest identified many differences between divergent strains and far fewer between genetically close strains, as expected. We also identified regions segregating between normotensive and hypertensive strains. Conclusions: We have employed a strategy for identifying a mechanism of BP regulation by analyzing the pathway involvement of multiple genes. Participation of multiple genes with the ERK/MAPK Signalling pathway warrants further investigation of the contribution of each gene and sequence variant, individually or in combination, to hypertension.
Journal of Hypertension 01/2012; 30:e321. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A pharmacogenomic approach was used to further localize the genetic region responsible for previously observed enhanced cardiovascular sensitivity to propofol in Dahl Salt Sensitive (SS) versus control Brown Norway (BN) rats.
Propofol infusion levels that decreased blood pressure by 50% were measured in BN.13(SS) rats (substitution of SS chromosome 13 into BN) and in five congenic (partial substitution) strains of SS.13(BN). The effect of superfused 2,6 diisopropylphenol on small mesenteric arterial vascular smooth muscle transmembrane potential was measured in congenic strains before and during superfusion with Rp-adenosine-3',5'-cyclic monophosphorothioate and 2.5 μM (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, inhibitors of protein kinase A and G, respectively. The genetic locus and potential role of the renin gene in mediating vascular smooth muscle sensitivity to propofol were determined in three selected subcongenic SS.BN¹³ strains.
A 30-32% smaller propofol infusion rate reduced blood pressure by 50% in BN.13(SS) compared with BN and the SS.13(BN) congenic containing an 80 BN gene substitution. Compared with the 80 BN gene-containing SS.13(BN) congenic, SS exhibited greater protein kinase A dependent vascular smooth muscle hyperpolarization in response to propofol. Using subcongenics, the increased propofol-induced cardiovascular sensitivity and hyperpolarization was further localized to an eight-gene region (containing the BN renin gene). Blockade of angiotensin receptors with losartan in this subcongenic increased propofol-induced hyperpolarization by threefold to that observed in SS.
Enhanced cardiovascular sensitivity to propofol in SS (compared with BN) is caused by an altered renin gene. Through modified second messenger function, this differentially regulates vascular smooth muscle contractile state and reduces vascular tone, thereby exacerbating cardiovascular depression by propofol.
[Show abstract][Hide abstract] ABSTRACT: Impaired regulation of renin in Dahl salt-sensitive rats (SS/JRHsdMcwi, SS) contributes to attenuated angiogenesis in this strain. This study examined angiogenic function and genomic structure of regions surrounding the renin gene using subcongenic strains of the SS and BN/NHsdMcwi (BN) rat to identify important genomic variations between SS and BN involved in angiogenesis. Three candidate regions on Chr 13 were studied: two congenic strains containing 0.89 and 2.62 Mb portions of BN Chr 13 that excluded the BN renin allele and a third strain that contained a 2.02 Mb overlapping region that included the BN renin allele. Angiogenesis induced by electrical stimulation of the tibialis anterior muscle was attenuated in the SS compared with the BN. Congenics carrying the SS renin allele had impaired angiogenesis, while strains carrying the BN renin allele had angiogenesis restored. The exception was a congenic including a region of BN genome 0.4 Mb distal to renin that restored both renin regulation and angiogenesis. This suggests that there is a distant regulatory element in the BN capable of restoring normal regulation of the SS renin allele. The importance of ANG II in the restored angiogenic response was demonstrated by blocking with losartan. Sequencing of the 4.05 Mb candidate region in SS and BN revealed a total of 8,850 SNPs and other sequence variants. An analysis of the genes and their variants in the region suggested a number of pathways that may explain the impaired regulation of renin and angiogenesis in the SS rat.