Renal cortical and medullary blood flow responses to altered NO ...

Am J Physiol Regul Integr Comp Physiol 299: R1449–R1455, 2010. First published September 29, 2010; doi:10.1152/ajpregu.00440.2010.

Innovative Methodology

Renal cortical and medullary blood flow responses to altered NO availability in humans Mads Damkjær,1 Manoucher Vafaee,2 Michael L. Møller,3 Poul Erik Braad,4 Henrik Petersen,4 Poul Flemming Høilund-Carlsen,4 and Peter Bie1 1

Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark; 2Center of Functionally Integrative Neuroscience, Aarhus University, Aarhus University Hospital, Aarhus, Denmark; 3Department of Nuclear Medicine, Frederiksberg Hospital, Frederiksberg, Denmark; and 4Department of Nuclear Medicine, Odense University Hospital, Odense, Denmark Submitted 6 July 2010; accepted in final form 27 September 2010

Damkjær M, Vafaee M, Møller ML, Braad PE, Petersen H, Høilund-Carlsen PF, Bie P. Renal cortical and medullary blood flow responses to altered NO availability in humans. Am J Physiol Regul Integr Comp Physiol 299: R1449 –R1455, 2010. First published September 29, 2010; doi:10.1152/ajpregu.00440.2010.—The objective of this study was to quantify regional renal blood flow in humans. In nine young volunteers on a controlled diet, the lower abdomen was CT-scanned, and regional renal blood flow was determined by positron emission tomography (PET) scanning using H215O as tracer. Measurements were performed at baseline, during constant intravenous infusion of nitric oxide (NO) donor glyceryl nitrate and after intravenous injection of NO synthase inhibitor N␻-monomethyl-Larginine (L-NMMA). Using the CT image, the kidney pole areas were delineated as volumes of interest (VOI). In the data analysis, tissue layers with a thickness of one voxel were eliminated stepwise from the external surface of the VOI (voxel peeling), and the blood flow subsequently was determined in each new, reduced VOI. Blood flow in the shrinking VOIs decreased as the number of cycles of voxel peeling increased. After 4 –5 cycles, blood flow was not reduced further by additional voxel peeling. This volume-insensitive flow was measured to be 2.30 ⫾ 0.17 ml·g tissue⫺1·min⫺1 during the control period; it increased during infusion of glyceryl nitrate to 2.97 ⫾ 0.18 ml·g tissue⫺1·min⫺1 (P ⬍ 0.05) and decreased after L-NMMA injection to 1.57 ⫾ 0.17 ml·g tissue⫺1·min⫺1 (P ⬍ 0.05). Cortical blood flow was 4.67 ⫾ 0.31 ml·g tissue⫺1·min⫺1 during control, unchanged by glyceryl nitrate, and decreased after L-NMMA [3.48 ⫾ 0.23 ml·(g·min)⫺1, P ⬍ 0.05]. PET/CT scanning allows identification of a renal medullary region in which the measured blood flow is 1) low, 2) independent of reduction in the VOI, and 3) reactive to changes in systemic NO supply. The technique seems to provide indices of renal medullary blood flow in humans. blood pressure; humans; renal medulla; renal perfusion; PET/CT; L-NMMA; glyceryl nitrate IN THE KIDNEY, BLOOD FLOW in the cortex and medulla is regulated independently (2, 7, 22). Only a small fraction (⬃10%) of total renal blood flow enters the renal medulla. However, the regulation of this flow is important because it seems to play a key role in the regulation of tubular function, sodium excretion, fluid volume control, and ultimately blood pressure regulation (2, 11, 24). Studies of renal medullary blood flow are complicated by the complex vascular anatomy of the region (29, 30). The medulla is perfused from efferent arterioles of the inner cortical or juxtamedullary nephrons. These vessels form the

Address for reprint requests and other correspondence: P. Bie, Institute of Molecular Medicine, Univ. of Southern Denmark, 21 J. B. Winsloews Vej, DK-5000 Odense, Denmark (e-mail: [email protected]). http://www.ajpregu.org

descending vasa recta. At the border between the inner and outer medulla, vascular bundles are formed containing both the descending vasa recta and ascending vasa recta. The descending vasa recta and ascending vasa recta are in close apposition favoring efficient equilibration by counter-current exchange. This arrangement helps to preserve the corticomedullary concentration gradients of sodium chloride (NaCl) and urea necessary for urinary concentration, but also influences the kinetics of flow tracers. Measurement of regional renal blood flow can be approached experimentally by several distinct methods. Video microscopic measurement of red blood cell velocity is a highly reliable method but limited to single surface vessels of the cortex of the surgically exposed kidney or to vasa recta of the surgically exposed papilla. Laser Doppler flowmetry has been used extensively in anesthetized as well as conscious animals, although absolute flows cannot be obtained. The advantages of the method are that repeated measurements can be performed in the same region and that the use of several probes allows measurements to be obtained simultaneously at different depths in the parenchyma (29). However, neither of these two methods can be used in humans, as they are highly invasive. Quantitative positron emission tomography (PET) is a functional imaging technique that among other things can provide estimates of regional blood flow noninvasively in selected organs by mathematical modeling of the kinetic behavior of a radiotracer, e.g., H215O. It is considered the method of choice for estimations in humans of regional cerebral blood flow (31) and regional myocardial blood flow (10). Only a few studies have been undertaken to apply this method to the quantification of overall renal blood flow in humans. These have shown that it is feasible to assess renal blood flow by PET/CT in humans and that dynamic images from the abdominal aorta can be used as arterial input function (1, 13, 18, 28). The latter is important as it reduces the invasiveness of the method since the need for arterial blood samples is eliminated. Measurements of cortical and medullary blood flow do not appear to be available. CT scans provide precise anatomical localization of structures and organs, in theory making identification of renal cortex and medulla possible. Nitric oxide (NO) synthases (NOS) are a family of enzymes that catalyze the production of the powerful vasodilator NO from L-arginine. Chronic intrarenal infusions of the NOS inhibitor N␻-nitro-L-arginine methyl ester cause selective reduction of renal medullary blood flow in rats (25, 27). Infusion of the NOS substrate L-arginine increases renal medullary blood flow in spontaneous hypertensive rats (21). Taken together,

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Innovative Methodology R1450

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these animal studies suggest that altered NO availability is particularly important in the medulla, but also contributes to regulation of cortical perfusion. In the present study, we have assessed blood flow in the medullary region of human subjects by PET/CT. Pharmacological manipulation of NO availability was attempted to produce positive and negative changes in renal medullary blood flow. The aim was to examine whether such changes in medullary blood flow could be detected by PET/CT. MATERIALS AND METHODS

Subjects Experiments were carried out in nine healthy male volunteers aged 22–31 yr weighing 79.6 ⫾ 7.3 kg (means ⫾ SD) with a body mass index of 23.4 ⫾ 2.1 kg/m⫺2. All subjects gave written informed consent. The study was approved by the local ethics committee (file no. S-2008060) and was performed in full compliance with the Declaration of Helsinki. Prior to the study, a clinical examination was performed in each subject. None of the subjects provided any medical history that suggested major illness or showed any signs of renal disease, arrhythmia, diabetes, or hypertension, and all participants denied use of any kind of medication for 2 wk prior to or during the study. All subjects were normotensive and had normal plasma sodium, potassium, and creatinine concentrations. Experimental Protocols Both the absolute values of regional renal blood flow and the changes in response to alterations in NO availability can be expected to be related to sodium balance. Therefore, 4 days prior to the investigation the subjects were placed on a standardized diet containing 150 mmol NaCl/ day prepared by the kitchen for special diets at the university hospital. The volunteers were allowed to drink water as desired. They agreed not to exercise on the day before the experiment. To assess compliance with the prescribed diet, 24-h urinary sodium excretion (0700 – 0700 h) was measured on the day prior to investigation. Before the experiment, the subjects were awakened at 0630 h. At 0645 h they consumed a light standardized breakfast. At 0700 h, urine samples were collected, and at 0710 h, they were taken to the laboratory. They were weighed and placed in the supine position on the PET/CT scanner bed for the duration of the experiment. An intravenous catheter (Venflon Pro 20Ga; Becton Dickinson, Helsingborg, Sweden) was placed in a dorsal vein of the left hand for infusions and bolus injections. Cardiac output and systemic vascular resistance were measured noninvasively by impedance cardiography (PhysioFlow PF-03; Manatec Biomedical, Macheren, France). Briefly, the impedance cardiograph measures stroke volume based on changes in thoracic impedance and heart rate (HR), allowing calculation of cardiac output. Autocalibration is based on the subject’s characteristics (age, height, body mass, and systolic/diastolic blood pressure at rest). The mean difference between the cardiac output values obtained in normal subjects at rest by the direct Fick and PhysioFlow methods has been found to be negligible (0.07 l/min) (6). Brachial blood pressure was measured every 10 min by an automatic oscillometric monitor (Colin Press-Mate BP-8800; ViCare Medical, Birkerod, Denmark). After 30 min of equilibration, PET/CT-measurements were carried out in 2D using a Discovery VCT PET/CT-scanner (GE Healthcare, Milwaukee, WI) (27). A low-dose helical CT-scan (64 slices) was acquired using a standard CT protocol with a transaxial scan field of view of 70 cm. Data was reconstructed with a field of view of 50 cm, matrix size of 512 ⫻ 512 (pixel size 0.98 mm) and a slice thickness of 3.75 mm using filtered back projection and noise filtered with a standard GE CT-filter. Subsequently, an injection of H215O was given intravenously (1,000 MBq in 5 ml of saline as a rapid bolus flushed in with 20 ml of saline), and the first dynamic 4-min PET-scan (baseline data) was performed. The dynamic PET scans (23 frames; 12 ⫻ 5 s ⫹ 6 ⫻ 10 s ⫹ 3 ⫻ 20 s ⫹ 2 ⫻ 30 s) were acquired with 47 images/frame spaced by 3.27 mm, AJP-Regul Integr Comp Physiol • VOL

and covering an axial field of view of 157 mm. The scan field of view was 70 cm. Attenuation correction was performed based on the CT-scan. The PET images were reconstructed as 128 ⫻ 128 matrices of 5.47 mm pixels transaxially, and a slice thickness of 3.75 mm, using the iterative method of ordered-subset expectation maximization (2 iterations, 20 subsets) and filtered with a 2D-Gaussian-filter (full width at half maximum at 4.29 mm), resulting in an isotropic resolution of ⬃5 mm. Next, an infusion of glyceryl nitrate (5 mg/ml Glycerylnitrat; Amgros, Copenhagen, Denmark) was started in a dose of 0.3 ␮g·kg⫺1·min⫺1. Due to considerable interindividual differences in sensitivity to glyceryl nitrate (8), the dose was increased stepwise until each subject had an increase in HR of some 10% (dose range 0.3– 0.9 ␮g·kg⫺1·min⫺1); HR was used as a surrogate marker of vasodilatation. Following a second injection of H215O, the second PET-scan (glyceryl nitrate) was performed. When this scan was completed, the glyceryl nitrate infusion was stopped. The pharmacological half-life of glyceryl nitrate is ⬃2 min. After the HR had returned to preinfusion levels, a bolus injection of N␻-monomethyl-Larginine (L-NMMA) was given intravenously (3 mg/kg, L-NMMA acetate; Clinalfa Basic, Bachem Distribution, Weil am Rhein, Germany). In other studies, this dose has been found to be an effective vasoconstrictor in humans (16). The final PET scan (L-NMMA) was performed 5 min after the L-NMMA injection. Side Effects of Medication During the infusion of glyceryl nitrate, seven of nine subjects reported unwanted side effects in the form of headache, tension in shoulders, and slight nausea. None of the subjects experienced any side effects after the L-NMMA bolus. Unscheduled termination due to side effects did not occur. Data Analysis The scanner software automatically coregisters the PET and CT scans. Before image processing, all scans were inspected to check alignment between the PET and CT images, see Fig. 1 for a representative image. Renal medulla. Data were analyzed stepwise as follows. Via the CT-scan image, the kidney poles were identified, and the longitudinal axis was defined as the line between them. Planes perpendicular to the longitudinal axis at points one-fourth of the interpolar distance from the poles defined the intrarenal limits of the original volumes of interest (OVOI). The OVOI was drawn using Display software (open-source program Display; Montreal Neurological Institute, McGill University, Montreal, Canada, http://www.bic.mni.mcgill.ca/⬃david/). Tissue layers with a thickness equivalent to one PET voxel (3.27 mm) were eliminated from the outer cortical part of the VOI in a stepwise fashion starting with the most superficial layer (voxel peeling), see Fig. 2. Following the removal of each layer, the blood flow of the remaining part of the VOI was calculated (see below). Renal cortex. With the use of the same procedure as above, the OVOI was defined. A tissue layer with a thickness equivalent to one voxel was eliminated from the outer cortical part of the OVOI, resulting in a reduced VOI. This was then subtracted from OVOI using Medical Imaging NetCDF (MINC) tools. With this approach, an outer cortical layer with a thickness of one voxel was identified (Fig. 3), and the blood flow in this layer was calculated. Processing of PET data. Assuming tracer clearance via the urine to be negligible, the blood flow was calculated based on the KetySchmidt one-compartment model (20). Curve fitting was performed by use of both a one-compartment and a two-compartment model. Results generated by use of the one-compartment model are reported because this procedure consistently gave the best fit. The fitting program yields the kinetic parameter K1, which is indicative of tracer clearance from blood to tissue; this was then corrected for an extraction fraction of 0.85 to obtain blood flow (15). The tracer concentration in the blood of the abdominal aorta was used as arterial input function. A small region of interest (⬃5 voxels) was defined in the 299 • DECEMBER 2010 •

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Fig. 1. Summed positron emission tomography (PET) image coregistered to CT-scan. Scale bar indicates counts per second in the PET image.

center of the aorta on the summed PET image where all of the 23 dynamic image frames were converted to one static image. This was done in all transaxial slices with the exception of the most cranial and most caudal slice, giving a total of 45 slices. This method has been shown to be a suitable alternative to arterial blood sampling (13, 19). Statistical Analysis

RESULTS

For all nine subjects, the rates of sodium excretion on the day prior to the experiment were similar, 150 ⫾ 12 mmol/24 h, indicating compliance with the dietary protocol. Systemic Hemodynamics

All values are presented as means ⫾ SE. Comparisons were performed by one-way ANOVA for repeated measures. In the case of significant differences, post hoc Dunnett’s tests were performed. Statistical calculations were performed with GraphPad Prism (GraphPad Software, San Diego, CA). Differences were considered significant at P ⬍ 0.05.

The main cardiovascular response parameter was HR. In the baseline setting this was stable at 63.2 ⫾ 3.3 beats/min. During the glyceryl nitrate infusion it increased 14 ⫾ 3% to 74.5 ⫾ 3.4 beats/min (P ⬍ 0.001). After the L-NMMA injection, HR

Fig. 2. Representative CT-image of the kidney from one subject. A: lower kidney pole region (red) defined as the original volume of interest (OVOI). B: superficial tissue layer with a thickness equivalent to one PET voxel (3.27 mm) was eliminated from the OVOI to obtain a new reduced VOI. C: subtraction of a tissue layer of one voxel was repeated to obtain a new further reduced VOI (OVOI minus 2 voxel layers). D: another voxel layer was eliminated to obtain a further reduced VOI (OVOI minus 3 voxel layers). This process was repeated as long as technically possible.

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Fig. 3. Representative CT-image of the kidney from one subject. A: lower kidney pole region (re) defined as OVOI. B: 1 voxel layer was subtracted to obtain the reduced VOI. C: reduced VOI was subtracted from the OVOI, leaving only the most superficial voxel layer.

decreased to 49.8 ⫾ 3.5 (P ⬍ 0.001) (Fig. 4A). Mean arterial blood pressure decreased by 8 ⫾ 2% (P ⬍ 0.01) on glyceryl nitrate and increased 8% (P ⬍ 0.01) on L-NMMA (Fig. 4B). Cardiac output was unchanged during glyceryl nitrate, and decreased 27 ⫾ 2.19% (P ⬍ 0.01) after L-NMMA (Fig. 4C). As expected, the infusion of glyceryl nitrate caused a decrease in systemic vascular resistance (10 ⫾ 4%, P ⬍ 0.05), and the injection of L-NMMA was followed by an increase (20 ⫾ 3%, P ⬍ 0.001) (Fig. 4D). Infusion of the NO-donor glyceryl nitrate caused significant systemic vasodilatation; the opposite effect was observed after bolus injection of the NOS-inhibitor L-NMMA.

Renal Medulla The initial blood flow in the OVOI, including cortex and medulla, was 4.39 ⫾ 0.18 ml·g tissue⫺1·min⫺1 (Fig. 5). The blood flow of the VOI decreased as the number of cycles of voxel peeling increased. After 4 –5 cycles of peeling, the flow in the reduced VOI became insensitive to additional peeling (Fig. 5). This volume-insensitive flow was 2.30 ⫾ 0.17 ml·g tissue⫺1·min⫺1 at baseline and increased to 2.97 ⫾ 0.18 ml·g tissue⫺1·min⫺1 (P ⬍ 0.05) during infusion of glyceryl nitrate. The bolus of L-NMMA caused a decrease to 1.57 ⫾ 0.17 ml·g tissue⫺1·min⫺1 (P ⬍ 0.05, Fig. 6A).

Fig. 4. Hemodynamic parameters during the study. Heart rate (A), mean arterial blood pressure (B), cardiac output (C), and systemic vascular resistance (D). L-NMMA, N␻-monomethyl-L-arginine. Statistically significant differences from baseline values: *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001.


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Fig. 5. Regional renal blood flow plotted as a function of the number of voxel layers peeled off of the OVOI. Statistically significant differences from the value in the OVOI: ***P ⬍ 0.001.

Renal Cortex In the outermost voxel layer of the kidney, the flow was 4.67 ⫾ 0.31 ml·g tissue⫺1·min⫺1 (Fig. 6B). It was unaltered during infusion of glyceryl nitrate (4.66 ⫾ 0.53 ml·g tissue⫺1·min⫺1). The bolus of L-NMMA was followed by a decrease to 3.48 ⫾ 0.23 ml·g tissue⫺1·min⫺1 (34% ⫾ 19, P ⬍ 0.05). DISCUSSION

The primary aim of the present study was to establish a method for measuring human renal cortical and medullary

Fig. 6. Regional renal blood flow at baseline and during changes in nitric oxide (NO) availability in the smallest VOI (OVOI minus 5 voxel layers) i.e., medullary blood flow (A) and in the most superficial voxel layer, i.e., cortical blood flow (B) cf. Fig. 3C. Statistically significant differences compared with baseline: *P ⬍ 0.05. AJP-Regul Integr Comp Physiol • VOL

perfusion. Apparently, we have attained this objective. However, any study attempting to measure renal medullary blood flow in humans is faced with two major problems. First, there is no other method to which the results can be compared, and second, the highly complex vascular anatomy, which provides excellent conditions for countercurrent exchange, raises questions about the validity of the simple algorithms traditionally used for calculation of flows. The validity of the present data is best assessed by simultaneous measurements carried out with the present method and another independent method capable of quantification of local blood flow, e.g., tissue perfusion by laser Doppler devices. Such a comparison is not without difficulties: 1) laser Doppler flowmetry does not provide absolute values of flow, but only relative changes (7, 24, 34); and 2) the method is invasive to an extent that makes it nonfeasible for use in humans. Nevertheless, laser Doppler flowmetry in an appropriate large experimental animal with a kidney structure similar to man, e.g., the pig, seems to be the preferable choice for validation. At present, we do not have access to facilities allowing for simultaneous renal laser Doppler flowmetry and PET/CT scanning in large experimental animals; therefore, this issue remains to be addressed by future experiments. In anesthetized pigs, comparison of total renal blood flow determination by ultrasound and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been performed recently (23). In this study, an ultrasound probe was placed on the renal vein through a laparotomy, and the animals were investigated while still anesthetized. After recording of morphological images, an intravascular contrast agent (gadofosveset trisodium) was administered prior to the determination of total and regional renal blood flow. The results show that total renal blood flow values can be estimated by DCE-MRI within a range of 60 to 170 ml·min⫺1·100 cm3 tissue⫺1 with an error of ⬍10%. Calculations of the regional blood flow values were based on the flow distribution assessed by DCE-MRI and the total renal blood flow obtained with the ultrasound probe. The renal medullary blood flow obtained with this method (⬃0.90 ml·min⫺1·cm3 tissue⫺1) were lower than those found in the present study (some 2.3 ml·min⫺1·cm3 tissue⫺1). However, a number of differences between the study of Ludemann et al. (23) and the present study may account for this discrepancy. Notably, Ludemann et al. used anesthetized, laparotomized pigs, an artificial contrast agent, and the combination of MRI

Fig. 7. Blood flow in each individual voxel layer. Layer 0 is the most superficial layer, whereas layer 5 is the smallest VOI (OVOI minus 5 voxel layers) i.e., medullary region. Layers 1 through 4 are those stepwise peeled off of the OVOI. 299 • DECEMBER 2010 •

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and ultrasound measurements. In contrast, we investigated conscious, healthy humans by use of labeled water and PET scanning. The interference of the vascular counter-current arrangement of the renal medulla is another potential problem. The question of diffusion, to a disturbing extent within the appropriate time frame of labeled water from the descending to the ascending vasa recta, is not easily addressed. In an organ without countercurrent structures, an analogous problem would arise in the case of shunting of blood from the arterial to the venous vessels (a-v shunting), thereby providing a mismatch between the arterial input function and the tracer activity curve within the specific tissue in question. In the present situation, tracer diffusion from descending to ascending vasa recta will affect the results in a way similar to a-v shunting, but here it is possible to address the issue by measuring the blood flow in the individual voxel layers. If significant shunting occurred, then the flow values in the outer medulla should be spuriously high, while the values of the remaining smaller VOIs would be falsely low. Therefore, the profile of the measured values of perfusion in individual voxel layers is interesting. Generally, if quantified in single shells with a thickness equivalent to one voxel, the tissue perfusion pattern was characterized by a continuous decrease from a high level of cortical flow to levels of much smaller flow in the medullary region (Fig. 7). This smooth decrease does not provide conclusive evidence, but is consistent with lack of significant effect of tracer diffusion. It may be argued that if the perfusion drops abruptly at the corticomedullary border, then the effects of sizeable tracer diffusion will be a smoothing of this transition, and therefore the measured flow pattern will be similar to that seen in Fig. 7. This issue seems worth addressing in future experiments by multiple, simultaneous measurements of local blood flow at various depths. However, this seems to necessitate the use of relatively large experimental animals in which several laser Doppler flow probes can be placed at various depths in the kidney without significant disturbance of organ function. Current notions of the mechanisms responsible for sodium balance involve changes in renal medullary blood flow (2, 7). Long-term arterial blood pressure regulation seems to be the result of multiple blood pressure feedback loops operating in parallel (3, 12). In one of these feedback loops, blood volume is the regulated variable. Blood volume is a monotone function of sodium intake (4, 9, 33), and the mechanisms controlling sodium homeostasis are important, therefore, for long-term control of arterial blood pressure. The present method seems to provide a tool by which the relationship between blood volume and renal medullary blood flow can be investigated in humans. Perspectives and Significance We have developed a new method for measurement of blood flow in a well-defined area in the medullary region of the kidney. Taking anatomical and functional evidence together, it is reasonable to assume that this tissue predominantly represents the renal medulla. Future studies should include validation of this method by simultaneous investigations of local renal blood flow by PET/CT and Doppler methods in experimental animals, e.g., in pigs as well as an attempt to include an anatomical definition of the renal medulla. Fully validated, the method could become a valuable instrument in the analysis of AJP-Regul Integr Comp Physiol • VOL

normal homeostasis, as well as the pathophysiology of diseases, possibly involving the renal medullary circulation, e.g., essential hypertension. Acutely, sodium excretion may be increased by an order of magnitude without any change in arterial pressure (26, 32, 35), and chronically, there is no clear-cut relationship between sodium intake and arterial blood pressure (9, 14, 17); with the present method, it is possible to address the role of renal medullary circulation in the regulation of sodium excretion without changes in arterial pressure. Furthermore, the possible changes in renal medullary flow associated with essential hypertension can be addressed. GRANTS Funding for the study was provided by the Danish Cardiovascular Research Academy, the Danish Heart Association, and the Elsa and Gudmund Jørgensen’s Foundation. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by any of the authors. REFERENCES 1. Alpert NM, Rabito CA, Correia DJ, Babich JW, Littman BH, Tompkins RG, Rubin NT, Rubin RH, Fischman AJ. Mapping of local renal blood flow with PET and H215O. J Nucl Med 43: 470 –475, 2002. 2. Bergstrom G, Evans RG. Mechanisms underlying the antihypertensive functions of the renal medulla. Acta Physiol Scand 181: 475–486, 2004. 3. Bie P, Damkjaer M. Renin secretion and total body sodium: pathways of integrative control. Clin Exp Pharmacol Physiol 37: e34 –e42, 2010. 4. Brown WJ Jr, Brown FK, Krishan I. Exchangeable sodium and blood volume in normotensive and hypertensive humans on high and low sodium intake. Circulation 43: 508 –519, 1971. 6. Charloux A, Lonsdorfer-Wolf E, Richard R, Lampert E, OswaldMammosser M, Mettauer B, Geny B, Lonsdorfer J. A new impedance cardiograph device for the non-invasive evaluation of cardiac output at rest and during exercise: comparison with the “direct” Fick method. Eur J Appl Physiol 82: 313–320, 2000. 7. Cowley AW Jr. Role of the renal medulla in volume and arterial pressure regulation. Am J Physiol Regul Integr Comp Physiol 273: R1–R15, 1997. 8. Curry SH, Aburawi SM. Analysis, disposition and pharmacokinetics of nitroglycerin. Biopharm Drug Dispos 6: 235–280, 1985. 9. Damgaard M, Gabrielsen A, Heer M, Warberg J, Bie P, Christensen NJ, Norsk P. Effects of sodium intake on cardiovascular variables in humans during posture changes and ambulatory conditions. Am J Physiol Regul Integr Comp Physiol 283: R1404 –R1411, 2002. 10. Dobrucki LW, Sinusas AJ. PET and SPECT in cardiovascular molecular imaging. Natl Rev Cardiol 7: 38 –47, 2010. 11. Eppel GA, Malpas SC, Denton KM, Evans RG. Neural control of renal medullary perfusion. Clin Exp Pharmacol Physiol 31: 387–396, 2004. 12. Fink GD. Hypothesis: the systemic circulation as a regulated free-market economy. A new approach for understanding the long-term control of blood pressure. Clin Exp Pharmacol Physiol 32: 377–383, 2005. 13. Germano G, Chen BC, Huang SC, Gambhir SS, Hoffman EJ, Phelps ME. Use of the abdominal aorta for arterial input function determination in hepatic and renal PET studies. J Nucl Med 33: 613–620, 1992. 14. Heer M, Frings-Meuthen P, Titze J, Boschmann M, Frisch S, Baecker N, Beck L. Increasing sodium intake from a previous low or high intake affects water. Br J Nutr 101: 1286 –1294, 2009. 15. Herscovitch P, Markham J, Raichle ME. Brain blood flow measured with intravenous H215O. I. Theory and error analysis. J Nucl Med 24: 782–789, 1983. 16. Hjorth Lassen L, Klingenberg Iversen H, Olesen J. A dose-response study of nitric oxide synthase inhibition in different vascular beds in man. Eur J Clin Pharmacol 59: 499 –505, 2003. 17. Hooper L, Bartlett C, Davey Smith G, Ebrahim S. Systematic review of long term effects of advice to reduce dietary salt in adults. BMJ 325: 628, 2002. 18. Juillard L, Janier MF, Fouque D, Cinotti L, Maakel N, Le Bars D, Barthez PY, Pozet N, Laville M. Dynamic renal blood flow measurement 299 • DECEMBER 2010 •

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