Pierre Dumas

Brown Norway Chromosome 13 Confers Protection From High Salt to Consomic Dahl S Rat

Consomic rats (SS.BN13), in which chromosome 13 from normotensive inbred Brown Norway rats from a colony maintained at the Medical College of Wisconsin (BN/Mcw) was introgressed into the background of Dahl salt-sensitive (SS/Mcw) rats, also maintained in a colony at the Medical College of Wisconsin, were bred. The present studies determined the mean arterial pressure (MAP) responses to salt and renal and peripheral vascular responses to norepinephrine and angiotensin II; 24-hour protein excretion and histological analyses were used to assess renal pathology in rats that received a high salt (4% NaCl) diet for 4 weeks. MAP of rats measured daily during the fourth week averaged 170±3.3 mm Hg in SS/Mcw rats, 119±2.1 mm Hg in SS.BN13 rats, and 103±1.3 mm Hg in BN/Mcw rats. After salt depletion, MAP fell an average of 27±4.5 mm Hg in SS/Mcw rats, 9±2.6 mm Hg in SS.BN13 rats, and 11±3.0 mm Hg in BN/Mcw rats. Protein excretion of SS/Mcw rats on a high salt diet averaged 189±30 mg/24 h, 63±18 mg/24 h in SS.BN13 rats, and 40±6.4 mg/24 h in BN/Mcw rats. Compared with SS.BN13 and BN/Mcw rats, SS/Mcw rats exhibited significantly greater increases of renal vascular resistance in response to intravenous norepinephrine and angiotensin II. Severe medullary interstitial fibrosis and tubular necrosis after a high salt diet were found consistently in SS/Mcw rat kidneys but were largely absent in the SS.BN13 and BN/Mcw rat kidneys. A similar degree of glomerular sclerosis was found in both SS/Mcw and SS.BN13 rats. In rats fed a 0.4% salt diet, the glomerular filtration rate of SS/Mcw rats was significantly less than that of BN/Mcw and SS.BN13 rats. These results reveal a powerful gene, or set of genes, within chromosome 13 of BN/Mcw rats that confers protection from the detrimental effects of high salt to the SS/Mcw rats.

Protection Against Necrosis but Not Apoptosis by Heat-Stress Proteins in Vascular Smooth Muscle Cells: Evidence for Distinct Modes of Cell Death

We have reported previously that cultured vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR) show higher proliferation and cell death than normotensive controls. In addition to protecting cells against death, heat stress proteins (HSPs) appear to play a role in cell proliferation. This investigation examines the involvement of HSP72 and HSP27 in altered SHR VSMC proliferation and death. We have performed detailed discriminatory analysis to characterize which type of VSMC death is induced by heat stress (HS) and serum deprivation. Serum deprivation induced apoptosis (caspase-3 cleavage and DNA laddering) and secondary necrosis, the 2 processes being a continuum of each other. In contrast, acute HS (46 degrees C, 30 minutes), which inhibited BN. lx and SHR VSMC proliferation by 2-fold, increased necrosis (by 5-fold and 2-fold, respectively) but not apoptosis. HSP72 and HSP27 expression evoked in VSMC by mild HS (44 degrees C, 15 minutes) 6 hours before acute HS prevented the inhibition of proliferation and induction of necrosis with no effect on serum deprivation-induced or staurosporine-induced apoptosis. This induced expression of HSP72 and HSP27 did not eliminate the higher basal proliferation, apoptosis, and necrosis of SHR VSMC compared with BN.lx VSMC, suggesting that these HSPs are not involved in altered SHR VSMC proliferation and death. Also, although apoptosis and necrosis may be a continuum, in VSMC the 2 processes may be distinguished by HS, in which only necrosis is prevented by prior HSP accumulation. This observation may be of use in designing strategies for cellular protection.

Workshop: excess growth and apoptosis: is hypertension a case of accelerated aging of cardiovascular cells?

Several groups including ours have demonstrated cardiac hyperplasia in neonates from genetically hypertensive rat strains. We have shown that similar problems exist in the kidney as well. More recently, we found that excessive heart and kidney weight is neonatally related to inhibition of apoptosis. Using recombinant inbred strains derived from a reciprocal cross between Brown Norway and spontaneously hypertensive rat progenitor strains, we mapped the inhibition of neonatal apoptosis to 2 distinct loci on chromosomes 1 (Myl 2) and 18 (Abrb 2). Positional candidate genes at these loci are being explored. These studies have also demonstrated that the loci determining kidney and heart weights in neonates are distinct from those determining increased organ weight in adults. The impact of blood pressure per se is also divergent because adult kidney weight is negatively correlated whereas heart weight is positively correlated with it. Analyses by extremes of low and high percentiles from fetal life to adulthood identified a single locus determining heart weight at Acaa on chromosome 8 in newborn (P =0.0003) and adult (P =0.016) rats. The Acaa region contains a DNA mismatch repair gene (hMLH1). The kinetics of neonatal growth through adulthood by prelabeling DNA with [3H]thymidine in pregnant mares showed that although the growth process is complex and nonlinear in the kidney of hypertensive rats, there is an increased turnover of cells, that is, reduced half-life of DNA. This observation is supported by the presence of shorter telomere fragments in kidneys of spontaneously hypertensive rats. These studies suggest that cardiovascular cells from hypertensive animals are subject to accelerated turnover, potentially leading to their accelerated aging.

Mapping of quantitative trait loci (QTL) of differential stress gene expression in rat recombinant inbred strains.

Objective Stress has been shown to be a major environmental contributor to cardiovascular diseases through its effects on blood pressure variability and cardiac function. The cellular stress response is characterized by the expression of specific heat stress genes (hsps), under the transcriptional control of heat shock transcription factors (HSTFs). The levels of hsp mRNA depend on the severity of the stress, with hstf1 acting as a stress sensor. The aim of this work was to evaluate the genetic contribution of the variability in hsp expression, and to identify its putative quantitative trait loci (QTL). Methods Twenty recombinant inbred rat strains (RIS) were studied. The animals underwent a standardized, identical 1 h immobilization stress in restraint cages, followed by 1 h of rest before sacrifice. Total RNA was extracted from the heart, kidneys and adrenals, and the mRNA levels of hsp27, hsp70, hsp84, hsp86 and hsp105 were measured. The strain distribution pattern (SDP) of hsp expression was correlated with that of 475 polymorphic markers distributed throughout the RIS genome. A polymorphism of rat hstf1 in RIS was used for its mapping in RIS. Results Despite an identical stress being applied to all strains, hsp expression showed up to a 12-fold gradient with little intra-strain variability, indicative of a strong genetic contribution to the trait. Heritability ranged from 50 to 77% for most hsp genes in the three target organs. The continuous SDP of stress gene expression indicated the polygenic nature of the trait. A common locus on chromosome 7 (at D7Cebrp187s3 marker) was consistently associated with all hsp expression in most of the organs [with a likelihood of odds (LOD) score of 3.0 for hsp27 expression]. We have mapped rat hstf1 on chromosome 7 at the same locus. Finally, the D4Mit19 marker was significantly associated with hsp84 expression in the heart (LOD score of 3.1). Conclusion Two loci were linked with the differential expression of HSPs in response to immobilization stress in target organs of RIS. The chromosome 7 locus unveiled for all HSPs could explain up to 42% of the observed interstrain variability of hsp levels in response to stress. We propose hstf1 as a positional candidate at this locus.

Contribution of Autosomal Loci and the Y Chromosome to the Stress Response in Rats

Stress is a critical contributor to cardiovascular diseases through its impact on blood pressure variability and cardiac function. Familial clustering of reactivity to stress has been demonstrated in human subjects, and some rodent models of hypertension are hyperresponsive to stress. Therefore, the present study was designed to uncover the genetic determinants of the stress response. We performed a total genome linkage search to identify the loci of the body temperature response to immobilization stress in a set of recombinant inbred strains (RIS) originating from reciprocal crosses of spontaneously hypertensive rats (SHR) with a normotensive Brown Norway Lx strain. Two quantitative trait loci (QTLs) were revealed on chromosomes (Chrs) 10 and 12 (logarithm of odds scores, 2.2 and 1. 3, respectively). The effects of these QTLs were enhanced by a high sodium diet (logarithm of odds scores, 4.0 and 3.3 for Chrs 10 and 12, respectively), which is suggestive of a salt-sensitive component for the phenotype. Congenics for Chr 10 confirmed both the QTL and the salt effect in RIS. Negatively associated loci were also identified on Chrs 8 and 11. Interaction between the loci of Chrs 10 and 12 was demonstrated, with the rat strains bearing SHR alleles at both loci having the highest thermal response to stress. Furthermore, the Y Chr of SHR origin enhanced the response to immobilization stress, as demonstrated in 2 independent models, RIS and Y Chr consomics. However, its full effect requires autosomes of the SHR strain. These findings provide the first evidence for the genetic determination of reactivity to stress with interactions between autosomal loci and between the Y and autosomal Chrs that contribute to the explanation of the 46% of variance in the stress response.

TA Repeat Variation, Npr1 Expression, and Blood Pressure. Impact of the Ace Locus

Abstract—The activity of the atrial natriuretic peptide receptor (Npr1) is altered in spontaneously hypertensive rats (SHR) in relation to its mRNA levels, suggesting abnormal transcriptional control in hypertension. A single-stranded conformational polymorphism caused by a repetitive dinucleotide segment of 10 TA in BN-Lx and of 40 TA in SHR was localized at position −943 relative to the transcription start site of the Npr1 gene, downstream of a putative cGMP-regulatory region, and was the only sequence difference noted between the two strains. Transient transfections of −1520 to −920 Npr1 promoter-SV40-luciferase fusion vector showed that the construct from BN-Lx stimulated the SV40 promoter, whereas that from SHR slightly inhibited it. In contrast to the BN-Lx construct, the activity of the SHR fragment was refractory to downregulation by atrial natriuretic peptide. Genotype-phenotype correlation studies in recombinant inbred strains (RIS) derived from BN-Lx and SHR crosses revealed significant correlations of the TA repeat with basal guanylyl cyclase activity and Npr1 mRNA levels. The correlations were heightened by a locus on chromosome 10 containing the Ace gene. The highest basal guanylyl cyclase activity and Npr1 mRNA values were found in RIS with both genes (Npr1/ Ace) of BN genotypes, whereas the lowest were recorded in RIS, with the SHR genotypes at both loci. This was inversely correlated with diastolic blood pressure in these strains. In conclusion, the longer TA repeat unit in the promoter of Npr1 of SHR, in tandem with a putative cGMP responsive element, regulates the transcription of the Npr1 gene with consequences on diastolic blood pressure.

Association of age-dependent height and bone mineral density decline with increased arterial stiffness and rate of fractures in hypertensive individuals

Objective: Hypertension and osteoporosis are age-related health risks differentially expressed in men and women. Here we have analysed their prevalence in a randomly selected cross-sectional cohort [CARTaGENE (CaG) of Quebec, Canada and explored their existing relationships along with height, arterial stiffness and bone fractures. Methods: The principal cohort CaG included 20 007 individuals of age 40–70 years. Participants were subjected to an extensive phenotyping and a questionnaire of medical history and habits. Results: We determined the differences in height of participants and their relation to hypertension status and sex in this cohort and validated it in two other cohorts (The Canadian Heart Health Study and a family cohort from the Saguenay Lac Saint-Jean, a region of Quebec). In all three cohorts, we found that at younger age individuals with hypertension are taller than normotensive individuals, but they have a shorter stature at an older age compared with normotensive individuals. In CaG, we observed that hypertension, low bone mineral density (BMD) and arterial stiffness are strongly associated with height when adjusted for antihypertensive medications (P < 0.0001). Fractures are the net outcome of low BMD, and a significant association is observed (odds ratio = 2.34, confidence interval = 2.12–2.57); this relation was stronger in hypertensive individuals compared with normotensive individuals particularly in younger hypertensive individuals. In addition, we observed that increased arterial stiffness was significantly correlated with a low BMD in both men and women at all ages. Conclusion: Shorter stature in elderly, low BMD and fractures correlated with increased arterial stiffness and hypertension. We propose that hypertension and osteoporosis share components of accelerated aging.

Identification and chromosomal localization of ecogenetic components of electrolyte excretion

Objective To determine to what extent urinary excretion of blood pressure-modulating electrolytes is genetically determined, and to identify their chromosomal localization. Design and methods Twenty-six rat recombinant inbred strains (RIS) originating from reciprocal crosses of normotensive Brown Norway (BN . Lx) and spontaneously hypertensive rats (SHR) were used. A pilot experiment on a subset of strains determined that fasting decreases the impact of environmental noise and increases that of heritability. Twenty-four-hour urinary collections were obtained from fasting rats aged 6–12 weeks (3–8 rats per strain). Sodium (Na), potassium (K) and calcium (Ca) excretions were measured, and the Na/K ratio calculated. These phenotypes served as quantitative traits for the search of quantitative trait loci (QTLs) by scanning the RIS genome that was mapped with 475 polymorphic markers. Results Constant Na intake resulted in a low heritability for Na excretion, reflecting the environmental impact (intake = excretion), whereas fasting revealed a gradient among RIS indicative of the genetic component of the traits. In the fasting state, a locus on chromosome 14 was found to be significantly associated with K excretion (Alb, P = 0.00002, r = −0.69, logarithm of the odds score (LOD) 3.9), whereas another locus on chromosome 10 (D10Cebrp97s5, P = 0.0003, r = −0.69, LOD 3.0) and one on chromosome 6 (D6Cebrp97s14, P = 0.0007, r = −0.65, LOD 1.9) were more significantly associated with Na excretion and the Na/K ratio respectively. The observed correlations were all negative for Na, K and Na/K, indicating a higher excretion of Na and K and a greater Na/K ratio in rats bearing BN.Lx alleles at these loci, i.e. salt retention in fasting SHR. These three loci accounted for 47–55% of variance of their associated trait, suggesting that they are the main genetic determinants for these phenotypes in basal fasting conditions. Rats bearing the Y chromosome of SHR origin had significantly higher K excretion that, in turn, led to a significantly lower Na/K ratio. Finally, a locus on chromosome 7 was linked to Ca excretion, explaining 46% of the trait variance (D7Mit10, LOD score 3.0). Conclusion RIS enabled us to determine QTLs for environmentally modulated traits such as Na, K and Ca excretions. We demonstrated that whereas urinary electrolytes are determined mainly by intake (environment) in a steady state, their excretion in an adaptive state (fasting) is predominantly genetically determined by distinct QTL on autosomes as well as the Y chromosome. Furthermore, the loci responsible for Na and K excretions act independently of the locus governing the relative excretion of Na/K. Thus, the salt-retaining aspects of some hypertensives may be, in large part, determined by genes responsible for renal excretion, the impact of which is predominant over the environment under acute challenge.

Radio-Telemetry in Biomedical Research - Radio-Telemetry Blood Pressure Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research

Radiotelemetry is employed in several fields to circumvent several issues: areas difficult or dangerous to access, monitoring of dangerous processes, need for secret monitoring. In biological sciences, telemetry is mainly useful because it decreases the observer bias and interference. In the field of medicine, the current research is mostly aimed at findings the cause and appropriate cures to common diseases. Common diseases are widely prevalent diseases for which we know only partially the causes and for which, as a consequence, we only propose treatments to alleviate the symptoms or their impacts on target organs. The common examples of such diseases to name a few are: diabetes, cancer(s), obesity, multiple sclerosis and hypertension, not to mention most of the psychiatric illnesses. They are characterized by a strong genetic component and a strong environmental influence since their prevalence is markedly influenced by age, diet, exercise or other environmental stressors. This important environmental modulation makes them more difficult to study. Therefore, our goal here will be to illustrate the challenge of studying environmentmodulated traits. With hypertension as an example, we will describe the use and benefits of employing radiotelemetry in hypertension research in order to be able to subtract the role of the environment or, conversely to quantify its impact on blood pressure. In the current postgenome era, with enough financial support and colleagues from around the world, it has never been easier to design and perform huge genome-wide association studies to try to unveil the genetic determinants of common diseases. Each month, hundreds of loci are reported that are associated with a higher prevalence of diseases and single nucleotide polymorphisms covering the entire genome are proposed to be in linkage with disease genes. We also know that very few of these proposed loci end up being truly associated with diseases in replication studies and we will present the current arguments pro and against this approach in the field of hypertension. This, we hope, will illustrate the point that we want to make in this chapter: in order to perform valid genome-wide association studies in human or genetic studies in animal models to uncover the genetic determinants of common diseases, it is essential to clearly define the studied phenotype(s) and to ensure that their measurements are performed accurately with the least amount of confounders or artefacts.

798 Hypertensive patients are more at risk of developing osteoporosis and having fractures due to their low bone mineral density

Introduction: Hypertension and osteoporosis are age-related health risks differentially expressed in men and women. Here we have analyzed their prevalence in a population-based cohort (Cartagene) of Quebec, Canada. This cohort, established in 2010-2011, is composed of 20 000 randomly selected subjects of 40 to 70 years old. The recruitment of the participants was done by the Québec Health Board. Results: Our analysis shows that 32% of the participants are hypertensive, 12.8% osteopenic and 0.6% osteoporotic. We observed that hypertensive participants have a lower bone mineral density than normotensives (p < 0.0001); thus increasing their risk of fractures, particularly in women. However, hypertensive men have also a higher risk of fractures and a lower bone mineral density than normotensive men (p = 0.0260). Moreover, we observed that the interaction between hypertension and the fat free mass was very significant when the bone mineral density was taken into consideration. The risk of fractures increased with a low fat free mass and a BMI < 25 Kg/m2 (p < 0.0001). Conclusion: In conclusion, we observed that not only hypertensive women but also hypertensive men who are usually not diagnosed nor treated for osteoporosis have a lower bone mineral density and more fractures than normotensives. In addition, our results indicate that fat free mass is an important factor for the maintenance of high bone mineral density, in both men and women.