The overlooked significance of plasma volume for successful adaptation to high altitude in Sherpa and Andean natives.

In contrast to Andean natives, high-altitude Tibetans present with a lower hemoglobin concentration that correlates with reproductive success and exercise capacity. Decades of physiological and genomic research have assumed that the lower hemoglobin concentration in Himalayan natives results from a blunted erythropoietic response to hypoxia (i.e., no increase in total hemoglobin mass). In contrast, herein we test the hypothesis that the lower hemoglobin concentration is the result of greater plasma volume, rather than an absence of increased hemoglobin production. We assessed hemoglobin mass, plasma volume and blood volume in lowlanders at sea level, lowlanders acclimatized to high altitude, Himalayan Sherpa, and Andean Quechua, and explored the functional relevance of volumetric hematological measures to exercise capacity. Hemoglobin mass was highest in Andeans, but also was elevated in Sherpa compared with lowlanders. Sherpa demonstrated a larger plasma volume than Andeans, resulting in a comparable total blood volume at a lower hemoglobin concentration. Hemoglobin mass was positively related to exercise capacity in lowlanders at sea level and in Sherpa at high altitude, but not in Andean natives. Collectively, our findings demonstrate a unique adaptation in Sherpa that reorientates attention away from hemoglobin concentration and toward a paradigm where hemoglobin mass and plasma volume may represent phenotypes with adaptive significance at high altitude.

Kidney-specific genetic deletion of both AMPK α-subunits causes salt and water wasting.

AMP-activated kinase (AMPK) controls cell energy homeostasis by modulating ATP synthesis and expenditure. In vitro studies have suggested AMPK may also control key elements of renal epithelial electrolyte transport but in vivo physiological confirmation is still insufficient. We studied sodium renal handling and extracellular volume regulation in mice with genetic deletion of AMPK catalytic subunits. AMPKα1 knockout (KO) mice exhibit normal renal sodium handling and a moderate antidiuretic state. This is accompanied by higher urinary aldosterone excretion rates and reduced blood pressure. Plasma volume, however, was found to be increased compared with wild-type mice. Thus blood volume is preserved despite a significantly lower hematocrit. The lack of a defect in renal function in AMPKα1 KO mice could be explained by a compensatory upregulation in AMPK α2-subunit. Therefore, we used the Cre-loxP system to knock down AMPKα2 expression in renal epithelial cells. Combining this approach with the systemic deletion of AMPKα1 we achieved reduced renal AMPK activity, accompanied by a shift to a moderate water- and salt-wasting phenotype. Thus we confirm the physiologically relevant role of AMPK in the kidney. Furthermore, our results indicate that in vivo AMPK activity stimulates renal sodium and water reabsorption.

Specificity of Compensatory Reserve and Tissue Oxygenation as Early Predictors of Tolerance to Progressive Reductions in Central Blood Volume.

We previously reported that measurements of muscle oxygen saturation (SmO2) and the compensatory reserve index (CRI) provided earlier indication of reduced central blood volume than standard vital signs (e.g., blood pressure, heart rate, arterial oxygen saturation). In the present study, we hypothesized that the CRI would provide greater sensitivity and specificity to detect progressive decrease in central circulating blood volume compared with SmO2. Continuous noninvasive measures of CRI (calculated from feature changes in the photoplethysmographic arterial waveforms) were collected from 55 healthy volunteer subjects before and during stepwise lower body negative pressure (LBNP) to the onset of hemodynamic decompensation. Near infrared spectroscopy was used on the forearm to obtain deep SmO2, hydrogen ion concentration ([H]), and hemoglobin volume (HbT; decreases reflect vasoconstriction). CRI decreased by 97% in a linear fashion across progressive blood volume loss, with no clinically significant alterations in vital signs. The receiver operating characteristic (ROC) area under the curve (AUC) for the CRI was 0.91, with a sensitivity of 0.87 and specificity of 0.80, when predicting decompensation at progressive levels of LBNP. In comparison, SmO2, [H], and HbT had significantly lower ROC AUC, sensitivity and specificity values for detecting the same outcome. Consistent with our hypothesis, CRI detected central hypovolemia with significantly greater specificity than measures of tissue metabolism. Single measurement of CRI may enable more accurate triage, while CRI monitoring may allow for earlier detection of casualty deterioration.

Disruption of the endothelin A receptor in the nephron causes mild fluid volume expansion.

BACKGROUND: Endothelin, via endothelin A receptors (ETA), exerts multiple pathologic effects that contribute to disease pathogenesis throughout the body. ETA antagonists ameliorate many experimental diseases and have been extensively utilized in clinical trials. The utility of ETA blockers has been greatly limited, however, by fluid retention, sometimes leading to heart failure or death. To begin to examine this issue, the effect of genetic disruption of ETA in the nephron on blood pressure and salt handling was determined. METHODS: Mice were generated with doxycycline-inducible nephron-specific ETA deletion using Pax8-rtTA and LC-1 transgenes on the background of homozygous loxP-flanked ETA alleles. Arterial pressure, Na metabolism and measures of body fluid volume status (hematocrit and impedance plethysmography) were assessed. RESULTS: Absence of nephron ETA did not alter arterial pressure whether mice were ingesting a normal or high Na diet. Nephron ETA disruption did not detectably affect 24 hr Na excretion or urine volume regardless of Na intake. However, mice with nephron ETA knockout that were fed a high Na diet had mild fluid retention as evidenced by an increase in body weight and a fall in hematocrit. CONCLUSIONS: Genetic deletion of nephron ETA causes very modest fluid retention that does not alter arterial pressure. Nephron ETA, under normal conditions, likely do not play a major role in regulation of Na excretion or systemic hemodynamics.

Genetic influences on survival time after severe hemorrhage in inbred rat strains.

To find a genetic basis for differential ability to survive severe hemorrhage, we previously showed eightfold differences in survival times among inbred rat strains. We assumed that rat strains had similar normalized blood volumes (NBV; ml/100 g body wt). As NBV might vary among strains and constitute one genetic variable affecting survival time to hemorrhage, in experiment 1 of the current studies we first measured total blood volumes and calculated NBV in specific inbred rat strains (Brown Norway/Medical College of Wisconsin, BN; Dark Agouti, DA; Fawn Hooded Hypertensive, FHH; Lewis, LEW; and Dahl Salt-Sensitive, SS) previously found to be divergent in survival time. NBV differed by 20% (P < 0.01; BN > SS > FHH = LEW = DA) and had a heritability (h(2)) of 0.56. Hence, differential survival times in our previously published study might reflect strain-dependent differences in NBV. Then studies were conducted wherein rats were catheterized and, ∼24 h later, 47% of their blood volume was removed; these rats were observed for a maximum of 4 h. In experiment 2, blood volumes were measured the day prior to hemorrhage. Percent survival and survival time did not differ among strains. To obviate possible confounding effects of blood volume determination, in experiment 3 the average NBV for each strain was used to determine hemorrhage volumes. Percent survival (P < 0.01) and survival times (P < 0.001) were different with DA demonstrating the best (62.5%, 190 ± 29 min) and BN the worst (0%, 52 ± 5 min) survival responses. These data indicate that both blood volume and survival time after hemorrhage in rats are heritable quantitative traits, and continue to suggest that genetic assessment of these phenotypes might lead to novel therapeutics to improve survival to hemorrhage.

Hyperaldosteronism, hypervolemia, and increased blood pressure in mice expressing defective APC.

Adenomatous polyposis coli (APC) fosters degradation of beta-catenin, a multifunctional protein upregulating the serum- and glucocorticoid-inducible kinase (SGK1). SGK1 regulates a wide variety of renal transport processes. The present study explored the possibility that APC influences renal function. To this end, metabolic cage experiments were performed in mice carrying a loss-of-function mutation in the APC gene (apc(Min/+)), their wild-type littermates (apc(+/+)), and apc(Min/+) mice lacking functional SGK1 (apc(Min/+)/sgk1(-/-)). As a result, mean body weight, food intake, fluid intake, salt appetite, urinary flow, as well as plasma Na(+) and K(+) concentrations were similar in apc(Min/+) mice, apc(+/+) mice, and apc(Min/+)/sgk1(-/-) mice. Glomerular filtration rate and absolute renal Na(+) excretion were decreased, and fractional urinary K(+) excretion was enhanced in apc(Min/+) mice. The antinatriuresis, but not the hypofiltration and kaliuresis was partially reversed by additional lack of SGK1. Plasma corticosterone and aldosterone concentrations were significantly enhanced in apc(Min/+) mice. While the plasma corticosterone concentration was similar in apc(+/+) mice and apc(Min/+)/sgk1(-/-) mice, plasma aldosterone was even higher in apc(Min/+)/sgk1(-/-) mice than in apc(Min/+) mice. The hyperaldosteronism of apc(Min/+) mice was paralleled by significantly elevated plasma volume and blood pressure. The experiments reveal an influence of defective APC on adrenal hormone release and renal function, effects partially but not completely explained by APC dependence of SGK1 expression.

The heart communicates with the endothelium through the guanylyl cyclase-A receptor: acute handling of intravascular volume in response to volume expansion.

Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion [EC GC-A knockout (KO) mice] exhibit significant chronic hypervolemic hypertension. The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to fluorescein isothiocyanate-labeled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nm) increased microvascular fluorescein isothiocyanate-BSA extravasation in control but not EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg x min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice, the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure of control mice but rapidly and significantly increased central venous pressure of EC GC-A KO mice. In cultured lung endothelial cells, ANP provoked cGMP-dependent protein kinase I-mediated phosphorylation of vasodilator-stimulated phosphoprotein. We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by cGMP-dependent protein kinase I-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.

Sodium transporters in the distal nephron and disease implications.

The post-macula densa segments of the renal tubule--that is, the distal convoluted tubule, connecting tubule, and collecting duct--play a central role in determining final urine sodium excretion. The major regulated sodium transporters and channels in these cell types include the thiazide-sensitive (Na-Cl) cotransporter (NCC), the epithelial sodium channel (ENaC), and Na-K-ATPase. Furthermore, although not involved in sodium reabsorption, the anion exchanger, pendrin, and the basolateral bumetanide-sensitive Na-K-2Cl cotransporter (NKCC1 or BSC2) have roles in blood-volume maintenance. Mutations in several of these major sodium transporters, channel subunits, and their regulatory proteins have been linked to human diseases such as Liddle's syndrome, Gitelman's syndrome, and Gordon's syndrome, emphasizing the need for appropriate regulation of sodium at these sites for maintenance of sodium balance and normotension.

Impact of physiological variables and genetic background on myocardial frequency-resistivity relations in the intact beating murine heart.

Conductance measurements for generation of an instantaneous left ventricular (LV) volume signal in the mouse are limited, because the volume signal is a combination of blood and LV muscle, and only the blood signal is desired. We have developed a conductance system that operates at two simultaneous frequencies to identify and remove the myocardial contribution to the instantaneous volume signal. This system is based on the observation that myocardial resistivity varies with frequency, whereas blood resistivity does not. For calculation of LV blood volume with the dual-frequency conductance system in mice, in vivo murine myocardial resistivity was measured and combined with an analytic approach. The goals of the present study were to identify and minimize the sources of error in the measurement of myocardial resistivity to enhance the accuracy of the dual-frequency conductance system. We extended these findings to a gene-altered mouse model to determine the impact of measured myocardial resistivity on the calculation of LV pressure-volume relations. We examined the impact of temperature, timing of the measurement during the cardiac cycle, breeding strain, anisotropy, and intrameasurement and interanimal variability on the measurement of intact murine myocardial resistivity. Applying this knowledge to diabetic and nondiabetic 11- and 20- to 24-wk-old mice, we demonstrated differences in myocardial resistivity at low frequencies, enhancement of LV systolic function at 11 wk and LV dilation at 20-24 wk, and histological and electron-microscopic studies demonstrating greater glycogen deposition in the diabetic mice. This study demonstrated the accurate technique of measuring myocardial resistivity and its impact on the determination of LV pressure-volume relations in gene-altered mice.

Quantitative trait loci for baseline white blood cell count, platelet count, and mean platelet volume.

A substantial genetic contribution to baseline peripheral blood counts has been established. We performed quantitative trait locus/loci (QTL) analyses to identify chromosome (Chr) regions harboring genes influencing the baseline white blood cell (WBC) count, platelet (Plt) count, and mean platelet volume (MPV) in F(2) intercrosses between NZW/LacJ, SM/J, and C57BLKS/J inbred mice. We identified six significant WBC QTL: Wbcq1 (peak LOD score at 38 cM, Chr 1), Wbcq2 (42 cM, Chr 3), Wbcq3 (0 cM, Chr 15), Wbcq4 (58 cM, Chr 1), Wbcq5 (82 cM, Chr 1), and Wbcq6 (8 cM, Chr 14). Three significant Plt QTL were identified: Pltq1 (24 cM, Chr 2), Pltq2 (36 cM, Chr 7), and Pltq3 (10 cM, Chr 12). Two significant MPV QTL were identified, Mpvq1 (62 cM, Chr 15) and Mpvq2 (44 cM, Chr 8). In total, the WBC QTL accounted for up to 31% of the total variance in baseline WBC count, while the Plt and MPV QTL accounted for up to 30% and 49% of the total variance, respectively. These analyses underscore the genetic complexity underlying these traits in normal populations and provide the basis for future studies to identify novel genes involved in the regulation of mammalian hematopoiesis.

Hypervolemic hypertension in mice with systemic inactivation of the (floxed) guanylyl cyclase-A gene by alphaMHC-Cre-mediated recombination.

To dissect the tissue-specific functions of atrial natriuretic peptide (ANP), we recently introduced loxP sites into the murine gene for its receptor, guanylyl cyclase-A (GC-A), by homologous recombination (tri-lox GC-A). For either smooth-muscle or cardiomyocyte-restricted deletion of GC-A, floxed GC-A mice were mated to transgenic mice expressing Cre-recombinase under the control of the smooth-muscle SM22 or the cardiac alphaMHC promoter. As shown in these studies, Cre-mediated recombination of the floxed GC-A gene fully inactivated GC-A function in a cell-restricted manner. In the present study we show that alphaMHC-Cre, but not SM22-Cre, with high frequency generates genomic recombinations of the floxed GC-A gene segments which were transmitted to the germline. Alleles with partial or complete deletions were readily recovered from the next generation, after segregation of the Cre-transgene. We took advantage of this strategy to generate a new mouse line with global, systemic deletion of GC-A. Doppler-echocardiographic and physiological studies in these mice demonstrate for the first time the tremendous impact of ANP/GC-A dysfunction on chronic blood volume homeostasis.

Regional cerebral blood volume reduction in transgenic mutant APP (V717F, K670N/M671L) mice.

Recent advance in nuclear magnetic resonance (NMR) microimaging has enabled in vivo cerebral blood volume (CBV) mapping with high spatial resolution. Using an intravascular susceptibility contrast agent and T(2)-weighted magnetic resonance imaging (MRI) on a 9.4T NMR microimager, the regional CBV was measured in mice as the transverse relaxation increase induced by the contrast agent. CBV maps in an Alzheimer's disease mouse model at resting state were obtained and examined. Four-month-old male transgenic mutant APP (V717F, K670N/M671L) mice (N = 10) and littermate wild-type controls (N = 12) were used. Regional analysis of the multi-slice CBV maps revealed statistically significant CBV reductions among the APP mice in cerebral cortex (-9.29%, P = 0.0002), hippocampus (-4.22%, P = 0.02), and thalamus (-5.21%, P = 0.03), indicating an early change of microvasculature in these selected regions. No significant difference was found in olfactory bulb, pons, midbrain, superior colliculus, medulla, and cerebellum.

Heritability of venous function in humans.

OBJECTIVE: Venous function contributes to the pathogenesis of thrombophlebitis, venous thrombosis, and possibly to arterial hypertension. Venous disease is presumably heritable; however, the genetic variance of venous function is unknown. METHODS AND RESULTS: We determined the heritability of venous function in 46 twin pairs (24 monozygotic, age 35+/-11 years, 14 men, 34 women; 22 dizygotic, age 30+/-8 years, 19 men, 25 women). After a resting phase in the supine position, we determined venous function in both legs by impedance plethysmography. Venous capacity was determined by a standardized protocol. In addition, we obtained venous pressure volume curves by slowly deflating a thigh cuff from 60 to 0 mm Hg. Venous compliance was determined as the steepest part of the venous pressure volume curve. Heritability was estimated using a path modeling approach. Unadjusted heritability was 0.6 (P<0.05) for venous capacity and 0.9 (P<0.05) for venous compliance. The heritability estimate for venous capacity decreased to 0.3 after adjustment for age, body mass index, and body fat. The heritability estimate for venous compliance remained essentially unchanged after adjustment for sex and age. CONCLUSIONS: We conclude that venous function is strongly influenced by genetic factors. The genes involved may influence venous disease states.

Functional organisation of central cardiovascular pathways: studies using c-fos gene expression.

Until about 10 years ago, knowledge of the functional organisation of the central pathways that subserve cardiovascular responses to homeostatic challenges and other stressors was based almost entirely on studies in anaesthetised animals. More recently, however, many studies have used the method of the expression of immediate early genes, particularly the c-fos gene, to identify populations of central neurons that are activated by such challenges in conscious animals. In this review we first consider the advantages and limitations of this method. Then, we discuss how the application of the method of immediate early gene expression, when used alone or in combination with other methods, has contributed to our understanding of the central mechanisms that regulate the autonomic and neuroendocrine response to various cardiovascular challenges (e.g., hypotension, hypoxia, hypovolemia, and other stressors) as they operate in the conscious state. In general, the results of studies of central cardiovascular pathways using immediate early gene expression are consistent with previous studies in anaesthetised animals, but in addition have revealed other previously unrecognised pathways that also contribute to cardiovascular regulation. Finally, we briefly consider recent evidence indicating that immediate early gene expression can modify the functional properties of central cardiovascular neurons, and the possible significance of this in producing long-term changes in the regulation of the cardiovascular system both in normal and pathological conditions.

An intron 4 gene polymorphism in endothelial cell nitric oxide synthase might modulate volume-dependent hypertension in patients on hemodialysis.

Nitric oxide (NO) has some relevance to the pathophysiology of hypertension. We examined the distribution of endothelial nitric oxide synthase (ecNOS4) gene polymorphism in patients with essential hypertension (n = 107) in comparison with healthy subjects (n = 362), and then studied the possibility that ecNOS4 gene polymorphism affects changes in blood pressure (BP) due to water load in hemodialysis patients. Though the NO metabolite (NOx) level in subjects with the a allele (31.2 +/- 1.76 micromol/l) was significantly lower than in those without the a allele (35.6 +/- 0.90 micromol/l) (p = 0.0263), we found no association between this gene polymorphism and essential hypertension. However, there still remains the possibility that the NO response to elevation of BP might be suppressed in patients with hypertension. In order to examine the association between ecNOS4 gene polymorphism and volume-dependent hypertension, 181 hemodialysis patients with complete anuria were recruited. Depending on the increase in mean BP (mm Hg) divided by percent increase in body weight (deltaBP/deltaBW), patients were divided into high responders (High-R: deltaBP/deltaBW >2.0 mm Hg; n = 90) and low responders (Low-R: deltaBP/deltaBW <2.0 mm Hg; n = 91). In the High-R group, the frequencies of a/a, b/a and b/b genotypes were 1.1, 32.1 and 66.8%, respectively, and in the Low-R group, these frequencies were 1.1, 9.4 and 89.5%, respectively. The relative risk for those in the High-R group conferred by the ecNOS4 a allele (b/a + a/a) was 3.64 (95% confidence intervals: 1.60-8.24, p = 0.0089). This study did not show a strong involvement of ecNOS4 gene polymorphism, at least in the basal NO production in patients with essential hypertension, however, it indicated that ecNOS4 gene polymorphism might modulate changes in BP due to water load in patients on hemodialysis, thus indicating that these polymorphisms may be involved in the pathophysiology of volume-dependent hypertension.

Lose salt and gain a friend! A tribute to Gerhard Giebisch.

In this little essay I describe recent advances in understanding the problem of salt sensitivity and salt resistance involved in the control of blood volume and blood pressure. Genetic evidence links the recently characterized epithelial sodium channel (ENaC) and the potassium channel (ROMK-1) to monogenic diseases in humans, characterized by a renal salt-losing syndrome. A loss of function mutations in ROMK-1 gene causes in some pedigrees the syndrome of Bartter, characterized by metabolic alkalosis and a severe salt-losing syndrome. A loss of function mutations in ENaC genes causes pseudohypoaldosteronism-type 1, characterized by hypovolaemia, hyperkaliaemia, metabolic acidosis and hypotension. ENaC and ROMK-1 are expressed in the apical membrane of principal cells of the cortical collecting duct and their role in Na/K balance is briefly reviewed.

Uterine mass and uterine blood volume in mice selected 21 generations for alternative criteria to increase litter size.

Lines of mice, selected for 21 generations using alternative criteria to increase litter size, were evaluated for uterine mass and uterine blood volume to help explain differences in uterine capacity. For this study, mice were sampled from Generation 27, the sixth generation after relaxation of selection. Mice came from all four criteria of selection (LS = selection on number born to unaltered females; IX = selection on index of ovulation rate and ova success; UT = selection on uterine capacity; and LC = unselected control) in each of three replicates (a total of 12 lines). Measurement was at one of two stages, either 3 d or 6 d of gestation. Matings were at 10 wk of age, and a total of 508 mice (17 to 26 per line-day of pregnancy subclass) were measured. The mean of the three selected groups exceeded the control in uterine mass (P < .001), uterine blood volume (P < .002), uterine mass/body mass (P < .03), and uterine blood volume/body mass (P < .04) but not in uterine blood volume/uterine mass. Greater uterine mass and concomitantly greater uterine blood volume may have been partly responsible for greater uterine capacity resulting from LS, IX, and UT selections.