Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging.

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

Dynamic genetic linkage of intermediate blood pressure phenotypes during postural adaptations in a founder population.

Blood pressure (BP) is a dynamic phenotype that varies rapidly to adjust to changing environmental conditions. Standing upright is a recent evolutionary trait, and genetic factors that influence postural adaptations may contribute to BP variability. We studied the effect of posture on the genetics of BP and intermediate BP phenotypes. We included 384 sib-pairs in 64 sib-ships from families ascertained by early-onset hypertension and dyslipidemia. Blood pressure, three hemodynamic and seven neuroendocrine intermediate BP phenotypes were measured with subjects lying supine and standing upright. The effect of posture on estimates of heritability and genetic covariance was investigated in full pedigrees. Linkage was conducted on 196 candidate genes by sib-pair analyses, and empirical estimates of significance were obtained. A permutation algorithm was implemented to study the postural effect on linkage. ADRA1A, APO, CAST, CORIN, CRHR1, EDNRB, FGF2, GC, GJA1, KCNB2, MMP3, NPY, NR3C2, PLN, TGFBR2, TNFRSF6, and TRHR showed evidence of linkage with any phenotype in the supine position and not upon standing, whereas AKR1B1, CD36, EDNRA, F5, MMP9, PKD2, PON1, PPARG, PPARGC1A, PRKCA, and RET were specifically linked to standing phenotypes. Genetic profiling was undertaken to show genetic interactions among intermediate BP phenotypes and genes specific to each posture. When investigators perform genetic studies exclusively on a single posture, important genetic components of BP are missed. Supine and standing BPs have distinct genetic signatures. Standardized maneuvers influence the results of genetic investigations into BP, thus reflecting its dynamic regulation.

Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension.

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

Genetic determinants on rat chromosome 6 modulate variation in the hypercapnic ventilatory response using consomic strains.

To understand the genetic basis of pathways involved in the control of breathing, a large scale, high-throughput study using chromosomal substitution strains of rats is underway. Eight new consomic rat stains (SS-2(BN), SS-4(BN), SS-6(BN), SS-7(BN), SS-8(BN), SS-11(BN), SS-12(BN), SS-14(BN), SS-Y(BN)), containing one homozygous BN/NHsdMcwi (BN) chromosome on a background of SS/JrHsdMcwi (SS), were created by PhysGen (http://pga.mcw.edu) Program for Genomic Applications. Male and female rats were studied using standard plethysmography under control conditions and during acute hypoxia (inspired oxygen fraction = 0.12) and hypercapnia (inspired CO(2) fraction = 0.07). The rats were also studied during treadmill exercise. Both male and female BN rats had a significantly lower ventilatory response during 7% CO(2) compared with SS rats of the same gender. SS-6(BN) female rats had a significantly reduced ventilatory response, similar to BN rats due primarily to a reduced tidal volume. Male SS-6(BN) rats had a significantly reduced tidal volume response to hypercapnia but a slightly increased frequency response during hypercapnia. Gene(s) on the Y chromosome may play a role in this increased frequency response in the male rats because the SS-Y(BN) hypercapnic ventilatory response involves a significantly increased frequency response. Several chromosomal substitutions slightly altered the ventilatory responses to hypoxia and exercise. However, genes on chromosomes 6 and Y of those studied are of primary importance in aspects of ventilatory control currently studied.

Consomic rat model systems for physiological genomics.

A consomic rat strain is one in which an entire chromosome is introgressed into the isogenic background of another inbred strain using marker-assisted selection. The development and physiological screening of two inbred consomic rat panels on two genetic backgrounds (44 strains) is well underway. Consomic strains enable one to assign traits and quantitative trait loci (QTL) to chromosomes by surveying the panel of strains with substituted chromosomes. They enable the rapid development of congenic strains over a narrow region and enable one to perform F2 linkage studies to positionally locate QTL on a single chromosome with a fixed genetic background. These rodent model systems overcome many of the problems encountered with segregating crosses where even if linkage is found, each individual in the cross is genetically unique and the combination of genes cannot be reproduced or studied in detail. For physiologists, consomics enable studies to be performed in a replicative or longitudinal manner to elucidate in greater detail the sequential expression of genes responsible for the observed phenotypes of these animals. They often provide the best available inbred control strains for physiological comparisons with the parental strains and they enable one to assess the impact of a causal gene region in a genome by allowing comparisons of the effect of replacement of a specific chromosome on a disease susceptible or a resistant genomic background. Consomic rat strains are proving to be a unique scientific resource that can greatly extend our understanding of genes and their role in the regulation of complex function and disease.

Reactive oxygen species and molecular regulation of renal oxygenation.

It has been known since the 1940s that a gradient of renal oxygenation exists in the kidney with the lowest PO2 in the renal inner medulla under physiological conditions. Due to a low PO2 milieu in the renal medulla, the cells in this region are at constant risk of hypoxic injury. Although numerous studies have shown that renal medullary cells adapt well to low PO2, the precise mechanism mediating this adaptive response remains poorly understood. Recently, hypoxia-induced molecular adaptation in mammalian tissues or cells has been studied extensively and many studies have indicated that the molecular regulation of gene expression is importantly involved. This paper focuses on the role of a transcription factor, hypoxia-inducible factor-1 (HIF-1)-mediated molecular adaptation and explores the physiological relevance of molecular activation of HIF-1 and its target genes in the renal medulla. Given that this HIF-1-mediated action is associated with local redox status, evidence is presented to indicate that reactive oxygen species (ROS), especially superoxide (O) is importantly involved in HIF-1-mediated molecular adaptation in renal medullary cells. O degrades HIF-1alpha, an HIF-1 subunit, by activating ubiquitin-proteasome and thereby decreases the transcriptional activation of many oxygen-sensitive genes. This action of O disturbs renal medullary adaptation to low PO2 and produces renal medullary dysfunction, resulting in sodium retention and hypertension. This report also provides evidence indicating the primary source of O, enzymatic pathways for O production and activating mechanism of O production in the kidney. It is concluded that HIF-1-mediated molecular adaptation to low PO2 is of importance in the regulation of renal medullary function and that ROS may target this HIF-1-mediated medullary adaptation to damage renal function.

Mapping baroreceptor function to genome: a mathematical modeling approach.

To gain information about the genetic basis of a complex disease such as hypertension, blood pressure averages are often obtained and used as phenotypes in genetic mapping studies. In contrast, direct measurements of physiological regulatory mechanisms are not often obtained, due in large part to the time and expense required. As a result, little information about the genetic basis of physiological controlling mechanisms is available. Such information is important for disease diagnosis and treatment. In this article, we use a mathematical model of blood pressure to derive phenotypes related to the baroreceptor reflex, a short-term controller of blood pressure. The phenotypes are then used in a quantitative trait loci (QTL) mapping study to identify a potential genetic basis of this controller.

A genomic-systems biology map for cardiovascular function.

With the draft sequence of the human genome available, there is a need to better define gene function in the context of systems biology. We studied 239 cardiovascular and renal phenotypes in 113 male rats derived from an F2 intercross and mapped 81 of these traits onto the genome. Aggregates of traits were identified on chromosomes 1, 2, 7, and 18. Systems biology was assessed by examining patterns of correlations ("physiological profiles") that can be used for gene hunting, mechanism-based physiological studies, and, with comparative genomics, translating these data to the human genome.

Heritability estimates of obesity measures in siblings with and without hypertension.

The goal of the present study was to evaluate mean values and heritability estimates of 3 global and 11 regional obesity measures in siblings with (HPT, n=209) or without (non-HPT, n=91) early-onset (age

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.

Evidence that reduced renal medullary nitric oxide synthase activity of dahl s rats enables small elevations of arginine vasopressin to produce sustained hypertension.

On the basis of observations supporting the functional importance of nitric oxide (NO) in the regulation of renal medullary function, and a reduced nitric oxide synthase (NOS) enzyme activity in the outer medulla of the Dahl salt-sensitive (SS/Mcw) rats, we hypothesized that these inbred rats would have reduced capacity to synthesize renal medullary NO. This reduced capacity would sensitize them to the hypertensive effects of small elevations of circulating arginine vasopressin (AVP). SS/Mcw and Brown Norway (BN/Mcw) rats with implanted arterial and venous catheters were fed a 0.4% salt diet and infused intravenously for 14 days with a subpressor dose of AVP (2 ng/kg per min). Mean arterial pressure (MAP) was measured 2 hours daily in unanesthetized rats maintained in their home cages. MAP in SS/Mcw rats increased during day 1 of AVP infusion from a control level of 127+/-0.9 mm Hg to an average of 147+/-1.6 mm Hg after 14 days. MAP did not return to control values during the 3 days after the end of AVP infusion. BN/Mcw rats showed no changes of MAP during 14 days of AVP infusion (90.4+/-0.6 mm Hg and 92.3+/-0.4 mm Hg). Northern blot analysis of renal tissue from vehicle (saline) -infused rats demonstrated that NOS I and NOS III mRNA expression was significantly less in SS/Mcw rats in the renal outer medulla compared with BN/Mcw rats. We conclude that small, normally subpressor elevations of plasma AVP can produce chronic hypertension in SS/Mcw rats and that this phenomenon is related to the reduced medullary NOS enzyme activity, which in turn reduces the AVP-stimulated NO synthesis.

Production and actions of superoxide in the renal medulla.

The present study characterized the biochemical pathways responsible for superoxide (O(2)(-.)) production in different regions of the rat kidney and determined the role of O(2)(-.)in the control of renal medullary blood flow (MBF) and renal function. By use of dihydroethidium/DNA fluorescence spectrometry with microtiter plates, the production of O(2)(-. )was monitored when tissue homogenate from different kidney regions was incubated with substrates for the major O(2)(-.)-producing enzymes, such as NADH/NADPH oxidase, xanthine oxidase, and mitochondrial respiratory chain enzymes. The production of O(2)(-. )via NADH oxidase was greater (P<0.05) in the renal cortex and outer medulla (OM) than in the papilla. The mitochondrial enzyme activity for O(2)(-.)production was higher (P<0.05) in the OM than in the cortex and papilla. Compared with NADH oxidase and mitochondrial enzymes, xanthine oxidase and NADPH oxidase produced much less O(2)(-. )in the kidney under this condition. Overall, the renal OM exhibited the greatest enzyme activities for O(2)(-.)production. In anesthetized rats, renal medullary interstitial infusion of a superoxide dismutase inhibitor, diethyldithiocarbamate, markedly decreased renal MBF and sodium excretion. Diethyldithiocarbamate (5 mg/kg per minute by renal medullary interstitial infusion [RI]) reduced the renal medullary laser-Doppler flow signal from 0.6+/-0.04 to 0.4+/-0.03 V, a reduction of 33%, and both urine flow and sodium excretion decreased by 49%. In contrast, a membrane-permeable superoxide dismutase mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (TEMPOL, 30 micromol/kg per minute RI) increased MBF and sodium excretion by 34% and 69%, respectively. These effects of TEMPOL on renal MBF and sodium excretion were not altered by pretreatment with N(G)-nitro-L-arginine methyl ester (10 microgram/kg per minute RI). We conclude that (1) renal medullary O(2)(-. )is primarily produced in the renal OM; (2) both NADH oxidase and mitochondrial enzymes are responsible for the O(2)(-.)production in this kidney region; and (3) O(2)(-. )exerts a tonic regulatory action on renal MBF.

Measurement of regional blood flow in the kidney using laser-Doppler flowmetry.

The study of regional blood flow in the kidney has been fraught with difficulties. Quantitation of perfusion of the inner cortex and medulla of the kidney has been especially challenging to measure because there is no way to directly visualize the large or small blood vessels in these areas. Furthermore, there has been a lack of reliable techniques to measure tissue blood flow in the kidney. In order to appreciate fully the technical demands of measuring changes in regional blood flow within the kidney, it is necessary to first understand the complex structure of the renal microcirculation.

Axial stretching of extremity artery induces reversible hyperpolarization of smooth muscle cell membrane in vivo.

Circumferential stretch due to increases in pressure induces vascular smooth muscle cell depolarization and contraction known as the myogenic response. The aim of this study was to determine the in vivo effects of axial-longitudinal stretch of the rat saphenous artery (SA) on smooth muscle membrane potential (Em) and on external diameter. Consecutive elongations of the SA were carried out from resting length (L0) in 10% increments up to 140% L0 while changes in membrane potential and diameter were determined in intact and de-endothelized vessels. Axial stretching resulted in a small initial depolarization at 120% of L0 followed by a progressive 20 to 33% hyperpolarizaion of vascular smooth muscle between 130% and 140% of L0. At 140%, an average maximal 10.6 mV reversible hyperpolarization was measured compared to -41.2 +/- 0.49 mV Em at 100% L0. De-endothelialization completely eliminated the hyperpolarization to axial stretching and augmented the reduction of diameter beyond 120% L0. These results indicate that arteries have a mechanism to protect them from vasospasm that could otherwise occur with movements of the extremities.

Tubulointerstitial injury and loss of nitric oxide synthases parallel the development of hypertension in the Dahl-SS rat.

OBJECTIVE: Alterations in renal nitric oxide (NO) are involved in the hypertension of the Dahl salt-sensitive (Dahl-SS) rat We sought to identify the kinetics and sites of expression of the major NO synthase (NOS) isoforms. DESIGN: The renal expression of the major NOS were examined in Dahl-SS and salt-resistant rats (Dahl-SR) while on a low salt (0.1% NaCl) diet at 3 and 9 weeks of age. METHODS: Renal biopsies from Dahl-SS and Dahl-SR rats were compared for evidence of renal injury and for alterations in expression of the NOS enzymes by quantitative immunohistochemistry. RESULTS: At 3 weeks of age Dahl-SS and Dahl-SR rats have normal renal histology and similar immunohistochemical expression of NOS1, -2, and -3. At 9 weeks Dahl-SS rats had significantly higher blood pressure than Dahl-SR rats (P< 0.005 ), and lower macula densa NOS1 (P< 0.05) and cortical and medullary NOS3 (P< 0.05). NOS2 was reduced in cortical tubules in biopsies showing severe tubulointerstitial damage, but was not significantly different between Dahl-SS and Dahl-SR groups as a whole. Dahl-SS rats also manifested glomerular and tubulointerstitial injury. Tubular expression of osteopontin (OPN), which is an inhibitor of NOS2, correlated with the systolic BP in individual Dahl-SS rats (r2 = 0.80, P < 0.0001 ). CONCLUSION: Tubulointerstitial injury and the loss of NOS occur after birth and parallel the development of hypertension. We suggest that the structural and functional changes that occur with renal injury in the Dahl-SS rat may contribute to the development of hypertension.

Genetically defined risk of salt sensitivity in an intercross of Brown Norway and Dahl S rats.

A genetic segregation analysis was performed to identify genes that cosegregate with arterial blood pressure traits reflective of salt sensitivity. A population of 113 F2 male rats was derived from an intercross of inbred SS/JrHsd/Mcw (Dahl salt-sensitive) and BN/SsN/Mcw (Brown Norway) rats. Rats were maintained on an 8% salt diet from the age of 9 to 13 wk, and arterial pressure was measured for 3 h daily during the 4th wk of high salt intake in unanesthetized rats using implanted arterial catheters. At the end of the 3rd day of high-salt pressure recordings, the arterial pressure response to salt depletion was determined 1.5 days following treatment with Lasix and a low-sodium (0. 4%) diet. A genome-wide scan using 265 polymorphic simple sequence length polymorphism (SSLP) markers found that seven arterial pressure phenotypes determined at different times and circumstances, and representing two distinct indexes of salt sensitivity, mapped to the same region of rat chromosome 18. The trait of salt sensitivity was strongly influenced by the presence of SS alleles in this region of chromosome 18, and those rats which were homozygote SS/SS exhibited a significantly greater reduction of mean arterial pressure following sodium depletion (29 +/- 2 mmHg) than homozygote BN/BN (17 +/- 3 mmHg) or heterozygotic (22 +/- 2 mmHg) rats. This region of rat chromosome 18 corresponds to the long arm of human chromosome 5 and a region of human chromosome 18 that has been linked to hypertension in humans. Given the unlikely chance of these different blood pressure traits mapping to the same region, we believe these data provide evidence that this region of rat chromosome 18 plays an important role in salt-induced hypertension.

alpha(2)-adrenergic receptor-mediated increase in NO production buffers renal medullary vasoconstriction.

The present study was designed to investigate the role of nitric oxide (NO) in modulating the adrenergic vasoconstrictor response of the renal medullary circulation. In anesthetized rats, intravenous infusion of norepinephrine (NE) at a subpressor dose of 0.1 microgram. kg(-1). min(-1) did not alter renal cortical (CBF) and medullary (MBF) blood flows measured by laser-Doppler flowmetry nor medullary tissue PO(2) (P(m)O(2)) as measured by a polarographic microelectrode. In the presence of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) in the renal medulla, intravenous infusion of NE significantly reduced MBF by 30% and P(m)O(2) by 37%. With the use of an in vivo microdialysis-oxyhemoglobin NO-trapping technique, we found that intravenous infusion of NE increased interstitial NO concentrations by 43% in the renal medulla. NE-stimulated elevations of tissue NO were completely blocked either by renal medullary interstitial infusion of L-NAME or the alpha(2)-antagonist rauwolscine (30 microgram. kg(-1). min(-1)). Concurrently, intavenous infusion of NE resulted in a significant reduction of MBF in the presence of rauwolscine. The alpha(1)-antagonist prazosin (10 microgram. kg(-1). min(-1) renal medullary interstitial infusion) did not reduce the NE-induced increase in NO production, and NE increased MBF in the presence of prazosin. Microdissection and RT-PCR analyses demonstrated that the vasa recta expressed the mRNA of alpha(2B)-adrenergic receptors and that medullary thick ascending limb and collecting duct expressed the mRNA of both alpha(2A)- and alpha(2B)-adrenergic receptors. These subtypes of alpha(2)-adrenergic receptors may mediate NE-induced NO production in the renal medulla. We conclude that the increase in medullary NO production associated with the activation of alpha(2)-adrenergic receptors counteracts the vasoconstrictor effects of NE in the renal medulla and may play an important role in maintaining a constancy of MBF and medullary oxygenation.

Role of tumor necrosis factor-alpha gene locus in obesity and obesity-associated hypertension in French Canadians.

Obesity represents a serious risk factor for the development of cardiovascular diseases, including hypertension. Segregation studies suggest that obesity and obesity-associated hypertension may share some genetic determinants. The results of the present candidate gene investigation suggest that in hypertensive pedigrees of French-Canadian origin, one such determinant is the tumor necrosis factor (TNF)-alpha gene locus. Gender-pooled quantitative sib-pair analysis demonstrated a significant effect of the gene locus on 3 global and 7 regional measures of obesity (P=0.05 to 0.0004). Gender-separate quantitative sib-pair analyses showed that the impact of the locus on obesity is most significant in the abdominal region in men and in the thigh region in women. Furthermore, the haplotype relative-risk test demonstrated a significant association between the TNF-alpha gene locus and both obesity (P=0.006) and obesity-associated hypertension (P=0.02). These effects were most significant in individuals with nonmorbid obesity. In conclusion, the results of linkage and association analyses suggest that in hypertensive pedigrees of French-Canadian origin, the TNF-alpha gene locus contributes to the determination of obesity and obesity-associated hypertension. In addition, the data indicate that gender modifies the effect of the locus on the regional distribution of body fat.