Reciprocal Regulation of Plasma Apelin and Vasopressin by Osmotic Stimuli

CLINICAL RESEARCH www.jasn.org

Michel Azizi,* Xavier Iturrioz,† Anne Blanchard,* Se´verine Peyrard,* Nadia De Mota,† Nicolas Chartrel,‡ Hubert Vaudry,‡ Pierre Corvol,§ and Catherine Llorens-Cortes†

*Assistance Publique Hoˆpitaux de Paris, Hoˆpital Europe´en Georges Pompidou, Universite´ Paris Descartes, Faculte´ de Me´decine, and INSERM, CIC 9201, †INSERM, Unit 691, Colle`ge de France, ‡INSERM, Unit 413, Universite´de Rouen, and §INSERM, Unit 36, Colle`ge de France, Paris, France

ABSTRACT Apelin is a neuropeptide that co-localizes with vasopressin (AVP) in magnocellular neurons and is involved in body fluid homeostasis. Osmotic stimuli have opposite effects on the regulation of apelin and AVP secretion in animal models, but whether this is true in humans is unknown. This study investigated the relationship among osmolality, apelin, and AVP in 10 healthy men after infusion of hypertonic saline or loading with water to increase and decrease plasma osmolality, respectively. Increasing plasma osmolality was accompanied by a parallel, linear increase in plasma AVP concentration and by a decrease in plasma apelin concentration. In contrast, decreasing plasma osmolality by water loading reduced plasma AVP concentration and rapidly increased plasma apelin concentration. These findings suggest that regulation of apelin secretion contributes to the maintenance of body fluid homeostasis.

J Am Soc Nephrol 19: 1015–1024, 2008. doi: 10.1681/ASN.2007070816

The osmotic pressure of body fluids is maintained (putative receptor protein related to the angioten- within a remarkably narrow range in healthy adults. sin receptor AT1).6,7 It is a 36–amino acid peptide Body fluid homeostasis depends on neuronal path- (apelin 36) derived from a single 77–amino acid ways bearing very sensitive osmoreceptors,1 located precursor, proapelin.6,8,9 Proapelin has a fully con- along the lamina terminalis, including the circum- served C-terminal 17–amino acid sequence, apelin ventricular organs, such as the subfornical organ 17 (K17F), including the pyroglutamyl form of ape- and the organum vasculosum of the lamina termi- lin 13 (pE13F). K17F and pE13F both are present in nalis as well as the median preoptic nucleus.2 The rat brain and plasma,10 and apelin 36 is present in subfornical organ and organum vasculosum of the testis and uterus.11 All peptides exhibit a high affin- lamina terminalis are neuronally interconnected ity for the human8,12,13 and the rat apelin recep- with each other as well as with the median preoptic tors.14 Apelin possesses various cardiovascular nucleus and the hypothalamic paraventricular and functions (for reviews,15–17). Apelin and its receptor supraoptic nuclei.3 These neuronal pathways con- have been detected in the endothelial cells of large vert small changes in osmolality into a neuronal sig- conduit arteries, coronary vessels, and the endocar- nal to neurons that influence sensations of thirst 2 and systemic arginine vasopressin (AVP) release, Received July 25, 2007. Accepted November 13, 2007 thereby adjusting the intake or output of water to 4,5 Published online ahead of print. Publication date available at counteract changes in solute concentration. www.jasn.org. A recently discovered peptide, apelin, may also Correspondence: Dr. Catherine Llorens-Cortes, INSERM, Unit play a major role in the maintenance of body fluid 691, Colle`ge de France, Paris, F-75005 France. Phone: 33-1-44- homeostasis. Apelin, initially isolated from bovine 27-16-63; Fax: 33-1-44-27-16-67; E-mail: c.llorens-cortes@ stomach extracts,6 is the endogenous ligand of the college-de-france.fr human orphan G protein–coupled receptor APJ Copyright ᮊ 2008 by the American Society of Nephrology

J Am Soc Nephrol 19: 1015–1024, 2008 ISSN : 1046-6673/1905-1015 1015 CLINICAL RESEARCH www.jasn.org dium of the right atrium.18–20 Apelin injection decreases BP in animals,9,21–23 via nitric oxide production.21 Apelin has a positive inotropic effect both on isolated perfused rat hearts ex vivo24 and on normal and postmyocardial infarction rat hearts in vivo.25 Apelin administration in mice reduces left ventricular preload and afterload and increases contractile reserve and cardiac output.26 Finally, apelin-deficient mice develop heart failure with aging.27 Apelin and its receptor both are widely distributed in the brain9,14,28,29 but are particularly abundant in the supraoptic nucleus and paraventricular nucleus, where they co-localize with AVP in magnocellular neurons.10,29–31 Intracerebro- ventricular injection of K17F inhibits the typical phasic fir- ing pattern of AVP neurons in lactating rats, resulting in decreased systemic AVP release and increased aqueous Figure 1. Reverse-phase HPLC analysis of apelin-IR in human diuresis.10 Moreover, water deprivation in rats while in- plasma. A human plasma sample was chromatographed on a creasing systemic AVP release and causing depletion of Vydac C4 column, and the immunoreactive material contained in hypothalamic AVP stores decreases plasma apelin concen- the HPLC fractions was quantified by RIA. This chromatogram is trations and results in hypothalamic accumulation of the representative of four independent experiments giving the following percentage of each apelin fragment: pE13F, 30 Ϯ 13%, peptide. Thus, the two peptides are conversely regulated K17F, 56 Ϯ 12%, and apelin 36, 14 Ϯ 7%. The dotted line to optimize AVP release into the blood circulation and indicates the acetonitrile gradient. The arrows indicate the elution 10 prevent additional water loss through the kidney. positions of the reference peptides. Whether such opposite regulation of apelin and AVP secretion in response to osmotic stimuli is found in humans remains unknown. and 8:00 p.m. with no significant changes associated with the The aim of this clinical investigation was to examine the 12-h overnight fluid and food restriction (Table 1). The me- relationship between plasma osmolality, plasma apelin, and dian (IQR) within-subject variability in plasma apelin concen- plasma AVP in healthy adults in various states of hydration. tration as assessed by testing plasma samples collected between Hydration was modified by administration of a hypertonic sa- 8:00 a.m. on day Ϫ1 to 9:00 a.m. on day 1 was 18.6% (13.3 to line infusion and by water loading to increase and decrease 23.6%) and that of plasma AVP concentration was 23.3% (18.2 plasma osmolality, respectively. to 36.8%). There was no significant difference in plasma apelin or in plasma AVP concentrations between the individuals assigned to the hypertonic saline infusion on day 1 (group 1) and those RESULTS assigned to the water loading test on day 1 (group 2) at any time point between 8:00 a.m. on day Ϫ1 and 9:00 a.m. on day 1 Characterization of the Molecular Forms of Apelin (Table 1). The plasma apelin/AVP ratio was also similar be- Present in Human Plasma tween the two groups (Table 1). There was no significant cor- Apelin immunoreactivity (apelin-IR) was resolved as two ma- relation between plasma apelin concentration and plasma AVP jor components eluting in fraction 28 and fractions 31 to 32 concentration in the absence of osmotic challenge (data not and a minor component eluting in fraction 40 (Figure 1). Cal- shown). Although plasma osmolality levels significantly dif- ibration of the C4 column with apelin fragments revealed that fered between the two groups (Table 1), probably because the prominent immunoreactive compounds co-eluted with group 1 and group 2 were studied sequentially at different pE13F and K17F, respectively, and that the apelin-IR present in times of the year (see the Concise Methods section), there was fraction 40 exhibited the same retention time as synthetic ape- no significant correlation between plasma apelin or plasma lin 36 (Figure 1). AVP concentrations and plasma osmolality in the absence of osmotic challenge (data not shown). Plasma Apelin Levels in Physiologic Unrestricted Water Intake Conditions on Day ؊1 Hypertonic Saline Infusion in Group 1 At 8:00 a.m. on day Ϫ1, the plasma apelin concentration in 10 The hypertonic saline infusion induced a smooth and linear Ϯ Ϯ healthy individuals was 477 167 fmol/ml; plasma osmolality increase in plasma osmolality from 288 1.6 mOsm/kg H2O Ϯ Ϯ (286 4 mOsm/kg H2O) and plasma AVP concentration (me- at baseline to 302 3.1 mOsm/kg H2O at the end of the infu- dian [interquartile range (IQR)] 1.8 [1.4 to 3.0] fmol/ml) both sion (P Ͻ 0.0001 versus baseline; Figure 2A) and in plasma were within the physiologic range. Plasma osmolality, apelin, sodium (Naϩ) concentration (P Ͻ 0.0001 versus baseline; Ta- AVP, and apelin/AVP ratio remained stable between 8:00 a.m. ble 2), associated with a parallel linear increase in plasma AVP

1016 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1015–1024, 2008 www.jasn.org CLINICAL RESEARCH

concentration from 2.6 (2.4 to 3.5) fmol/ml at baseline to 10.0

1.38 (8.9 to 11.3) fmol/ml at the end of the infusion (P Ͻ 0.0001 2 2 2 83 ϭ 158 124 Ϯ Ϯ Ϯ versus baseline; Figure 2B). Ϯ Ϯ Ϯ 1,8 F There was a significant and marked decrease in hematocrit 335 (P ϭ 0.0017), total protein concentration (P ϭ 0.0003), and ͓ NS ͔ , plasma active renin (P ϭ 0.0019) and aldosterone concentra- 4.48 tions (P ϭ 0.0198) associated with an increase in the estimated ϭ Ϯ 1,8 extracellular fluid volumes (ECFV; 17.4 1.6%; Table 2). F 3 286 1 288 4 285 99 451 70 567 69 There was a highly significant positive linear correlation be- Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ tween plasma AVP concentration and plasma osmolality be- ϭ ϭ Ͻ

363 tween baseline and time 120 min (r 0.91, n 35, P 0.0001; Figure 3A), and the extrapolated median osmotic threshold for

AVP secretion was 283.1 mOsm/kg H2O (range 278.4 to 285 mOsm/kg H2O). The time course of plasma apelin concentration was bipha- 2 287 2 288 2 286 129 433 127 503 117 sic. During the first 80 min of the hypertonic saline infusion, Ϯ Ϯ Ϯ Ϫ 1 (8:00 a.m.) to day 1 (9:00 a.m.) in 10 Ϯ Ϯ Ϯ the plasma apelin concentration decreased linearly from 567 Ϯ

415 124 fmol/ml at baseline to 326 Ϯ 111 fmol/ml (P Ͻ 0.01 versus Ϫ 1 and 9:00 a.m. on day 1 (ANOVA, Ϫ Ϫ 0.02). Group 1: subjects receiving the hypertonic saline infusion on baseline; median change from baseline [IQR] 43.8% [ 47.2

ϭ to Ϫ20.4]; Figure 2C), contrary to the plasma AVP concentra- P Time

8, tion. Simple linear regression of plasma apelin and plasma os- ϭ 2 284 3 285 1 283 molality was thus applied to each individual infusion of hyper- 90 472 46 528 1,8 118 F Ϯ Ϯ Ϯ Ϯ Ϯ

Ϯ tonic saline between 0 and 80 min, the linear part of the decrease in plasma apelin with time. There was a highly significant negative linear correlation between plasma apelin concentration and plasma osmolality for the period 0 to 80 min (r ϭϪ0.70, n ϭ 25, P ϭ 0.0004; Figure 3B). There was also a significant negative linear correlation between plasma apelin 3 287 1 286 3 287 88 384 133 414 134 444 concentration and plasma AVP concentration for the period 0 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ to 80 min (r ϭϪ0.52, n ϭ 25, P ϭ 0.015; Figure 3C); however, after adjustment for plasma osmolality, the relationship between plasma apelin concentration and plasma AVP concentration was no longer significant (r ϭϪ0.31, n ϭ 25, P ϭ 0.3806; data not shown). This indicates that the correlation 4 286 2 288 3 284 between plasma apelin and AVP concentration observed dur- 167 467 196 541 101 393 Ϯ Ϯ Ϯ ing the first 80 min of the hypertonic saline infusion was driven Ϯ Ϯ Ϯ by the plasma osmolality. 8:00 a.m. 12:00 p.m. 4:00 p.m. 8:00 p.m. 12:00 a.m. 9:00 a.m. 1.8 (1.4 to 3.0) 2.3 (1.9 to 3.0) 1.9 (1.3 to 2.9) 2.2 (1.5 to 3.2) 2.4 (2.2 to 3.4) 2.3 (1.9 to 3.5) 3.0 (1.9 to 3.0) 2.7 (2.2 to 4.2) 2.5 (1.8 to 2.9) 1.9 (1.7 to 3.2) 3.2 (2.4 to 3.4) 2.6 (2.4 to 3.5) 1.5 (1.0 to 1.7) 1.9 (1.9 to 2.4) 1.3 (1.2 to 2.1) 2.4 (1.4 to 3.1) 2.0 (1.6 to 3.0)During 1.9 (1.9 to 2.1) the last 40 min of the hypertonic saline infusion, 233 (188 to 265) 156 (146 to 238) 223 (160 to 257) 202 (161 to 271) 169 (159 to 188) 192 (144 to 202) 212 (189 to 255) 152 (136 to 238) 196 (171 to 250) 229 (222 to 271) 165 (159 to 183) 196 (193 to 202) 265 (177 to 424) 157 (154 to 183) 251 (160 to 311) 172 (161 to 182)plasma 178 (127 to 207) apelin 144 (121 to 189) concentration increased from 326 Ϯ 111 fmol/ml at 80 min to 458 Ϯ 136 fmol/ml at 120 min (P ϭ 0.4961 versus baseline; Figure 2C). The plasma apelin/AVP ra- ͓ IQR ͔ ) SD) tio decreased massively from 196 (193 to 202) fmol/fmol at Ϯ baseline to 47 (30 to 50) fmol/fmol at time 80 min (P Ͻ 0.0001 versus baseline) and remained at a low value up to 120 min (52 Ͻ

SD) [46 to 52] fmol/fmol; P 0.0001 versus baseline; Figure 4). O; mean 2 Ϯ ͓ IQR ͔ ) To search for an explanation for the rise in plasma apelin a after its initial decrease, we used the individual equations of the regression line between plasma apelin and plasma osmolality 10) 478 10) 286 10) 10) Parameter ϭ ϭ ϭ ϭ

͓ NS ͔ , respectively). Plasma osmolality levels differed significantly between the two groups (ANOVA, between baseline and time 80 min to predict a curve for plasma 5) 552 5) 403 apelin between time 0 and time 120 min if plasma osmolality 5) 288 5) 284 5) 5) 5) 5) 0.29 ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ

ϭ was the sole determinant factor influencing plasma apelin lev- Plasma apelin, osmolality, and AVP levels in physiologic unrestricted water intake conditions from day

1,8 els. The predicted curve was plotted and compared with the F observed curve (Figure 5A). The two curves diverged from time point 80 min to time point 120 min (end of the infusion). all participants ( n all participants ( n group 1 ( n group 1 ( n group 2 ( n group 2 ( n all participants ( n group 1 ( n group 2 ( n all participants ( n group 1 ( n group 2 ( n Plasma apelin, AVP, and apelin/AVP ratio did not significantly differ between the two groups at any time point between 8:00 a.m. on day healthy male participants ͓ NS ͔ , and Table 1. a day 1. Group 2: subjects receiving the water load on day 1. Plasma apelin (fmol/ml; mean Plasma AVP (fmol/ml; median Plasma apelin/AVP ratio (fmol/fmol; median Plasma osmolality (mOsm/kg H We then plotted the absolute difference between the observed

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and the predicted apelin values at each time point (Figure 5B). From 80 min to the end of infusion, the difference between the observed and the predicted apelin values increased linearly. We then superimposed the time course of the relative changes in estimated ECFV from time 0 to time 120 min (Figure 5B). The observed plasma apelin concentration started to diverge from the predicted value (i.e., the difference diverged from 0) when the estimated ECFV had increased by approximately 12 to 15%. The expansion in the estimated ECFV had no apparent effect on plasma AVP concentrations (see Figure 2B).

Water Loading in Group 2 Plasma osmolality decreased rapidly and significantly from Ϯ baseline (284.6 1.8 mOsm/kg H2O) to a nadir at 60 min Ϫ (change from baseline 4.0 mOsm/kg H2O; 95% confidence Ϫ Ϫ ϭ interval 7.0 to 1.0 mOsm/kg H2O; P 0.0076) then pro- gressively increased to reach the baseline value at 240 min (Fig- ure 6A). The plasma Naϩ concentration decreased simulta- neously (P ϭ 0.0033 versus baseline; Table 2). There was a parallel significant decrease in plasma AVP concentration from 1.9 (1.8 to 2.1) fmol/ml at baseline to a nadir of 1.5 (1.4 to 1.7) fmol/ml (P ϭ 0.0139 versus baseline; median variation from baseline of Ϫ30.4% [IQR Ϫ45 to Ϫ28.6]) followed by a slight increase by 240 min (Figure 6B). Water loading had no significant effect on hematocrit or total protein, plasma active renin, and aldosterone concentrations, indicating no significant change in estimated ECFV (Table 2). In contrast to plasma AVP, the plasma apelin concentration increased from 335 Ϯ 83 to 527 Ϯ 187 fmol/ml (P ϭ 0.0047 versus baseline; median variation from baseline IQR 73.9% 23.4 to 74.5) immediately after water loading (time 30 min; Figure 6C); it then remained stable at approximately 530 fmol/ml for 210 min (time 240 min). The plasma apelin/AVP ratio increased massively from 144 (121 to 189) fmol/fmol at baseline to 424 (222 to 475) fmol/fmol immediately after water loading (P ϭ 0.0007 versus baseline; Figure 4); it then remained stable at approximately 300 fmol/fmol for 210 min (time 240 min).

DISCUSSION

The main finding of this study is that acute osmotic stimuli induce opposite regulation of plasma apelin and AVP concentrations in humans, as previously reported in rodents.10,31 Ape- lin-IR was previously detected in human plasma using com- mercially available apelin-36 RIA or apelin-12 EIA kits,32–36 but the data revealed an important disparity in the concentra-

and IQR. The ANOVA for repeated measurements over time on day 1 with a modeling covariance structure within subjects was signifi- Figure 2. Effects of a 2-h hypertonic saline infusion (0.85 M, 0.06 cant for all three variables. Pair-wise comparisons were tested using ml/kg per min) on plasma osmolality (A) and plasma AVP (B) and the Dunnett adjustment procedure for multiple tests comparing all apelin (C) concentrations. Plasma osmolality and apelin are ex- time points with baseline level at 9:00 a.m. on day 1. *P Ͻ 0.05; pressed as means Ϯ SEM. Plasma AVP is expressed as median **P Ͻ 0.01; ***P Ͻ 0.001 versus baseline (9:00 a.m. on day 1).

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Table 2. Hormonal, electrolyte, hematocrit, and total protein changes during hypertonic saline infusion and water loadinga Hypertonic Saline Infusion Water Loading Variables Time 0 Time 120 Time 0 Time 240 Plasma sodium (mmol/L; mean Ϯ SD) 138.8 Ϯ 0.9 147.4 Ϯ 0.9 137.0 Ϯ 1.2 136.5 Ϯ 1.9 Hematocrit (%; mean Ϯ SD) 42.9 Ϯ 2.4 38.6 Ϯ 3.0 43.4 Ϯ 2.1 44.7 Ϯ 3.0 Total proteins (g/L; mean Ϯ SD) 69.2 Ϯ 2.7 59.4 Ϯ 2.7 69.0 Ϯ 3.3 70.5 Ϯ 2.4 Plasma active renin (pg/ml; mean Ϯ SD) 35.8 Ϯ 8.4 17.4 Ϯ 3.2 22.0 Ϯ 6.4 24.0 Ϯ 8.0b Plasma aldosterone (pg/ml; median ͓IQR͔) 88 (79 to 145) 50 (33 to 52) 114 (84 to 117) 77 (69 to 120)b Cumulative Na input (mmol; mean Ϯ SD)c – 465 Ϯ 36 – 0 Cumulative Na output (mmol; mean Ϯ SD)c –53Ϯ 28 – 29 Ϯ 13 Cumulative water input (ml; mean Ϯ SD)c – 550 Ϯ 37 – 1546 Ϯ 68 Cumulative water output (ml; mean Ϯ SD)c – 255 Ϯ 106 – 1554 Ϯ 327 ECFV (L; mean Ϯ SD) 16.0 Ϯ 0.9 18.8 Ϯ 1.1 16.1 Ϯ 0.4 15.9 Ϯ 0.3 ECFV changes (%; mean Ϯ SD)d – 17.4 Ϯ 1.6 – Ϫ1.3 Ϯ 0.6 aThe hypertonic saline infusion caused a significant and marked decrease in hematocrit (P ϭ 0.0017), total protein concentration (P ϭ 0.0003), and plasma active renin (P ϭ 0.0019) and aldosterone (P ϭ 0.0198) concentrations. Water loading had no significant effect on hematocrit or total protein, plasma active renin, and aldosterone concentrations. bPlasma active renin and aldosterone were measured at time 180. cCumulative Na and water input/output are cumulative over 120 min for the hypertonic saline infusion or over 240 min for the water loading test. ϩ ϩ dECFV and ECFV changes (%) were estimated according to the following formula: Variation in ECFV (% of initial volume) ϭ͓(final P͓Na ͔)/net Na balance͔/ ϩ ϩ ϩ initial ECFV ϫ 100, with initial ECFV (L) ϭ͓(8116.6 ϫ BSA) Ϫ 28.2͔/1000, where BSA is body surface area and net Na balance (mmol/l) ϭ Na input Ϫ Na output. tions of apelin circulating in plasma of healthy individuals the hypertonic saline infusion was a potent physiologic stimu- (from 10 to 1000 fmol/ml).32–36 In addition, circulating apelin lus for AVP release. The median threshold of AVP release was in human plasma has only been analyzed by gel filtration,37 a within the expected physiologic range, consistent with previ- method that does not allow accurate characterization of the ous reports.4,5 Contrary to the changes in plasma AVP, the molecular forms of immunoreactive apelin. Here, we first linear increase in plasma osmolality had the opposite effect on identified the molecular forms of apelin present in human plasma apelin, the concentration of which declined linearly by plasma in vivo by combining HPLC analysis with RIA detec- 43.8% (20.4 to 47.2%) 80 min after the start of the infusion; tion. We showed for the first time that K17F and pE13F are the during this period, the plasma apelin concentration was highly predominant forms of apelin present in human plasma and significantly and negatively correlated to plasma osmolality that the concentration of apelin 36 is much lower, consistent and to plasma AVP concentration. The initial decrease in with previous findings in rodents.10 plasma apelin concentration during the first 80 min of the Using this assay, the physiologic concentrations of plasma hypertonic saline infusion was followed by a slight increase in apelin in 10 healthy lean male individuals on a normal sodium plasma apelin concentration during the last 40 min. Our data diet were 477 Ϯ 167 fmol/ml on day Ϫ1 at baseline (8:00 a.m.) suggest that the rise in the plasma apelin concentration after its after an overnight fast. On a molar basis, plasma apelin con- initial decrease may be triggered by the infusion-induced in- centration was physiologically 200 to 250 times higher than crease in ECFV (17.4% above its baseline) as also reflected by plasma AVP concentration (plasma apelin/AVP ratio: 233 the significant and marked decreases in hematocrit, total pro- [188 to 265] fmol/fmol) in the absence of any osmotic chal- tein, and plasma active renin, and aldosterone concentrations. lenge. This may reflect the multiple sources of circulating ape- In our study, this ECFV expansion had no detectable effect on lin. Circulating apelin originates at least in part from magno- plasma AVP concentration, although changes in pressure vol- cellular neurons10 but may also be produced by the ume are potent nonosmotic stimuli for AVP secretion.5,40 gastrointestinal tract,38 adipocytes,34 and vascular and endo- Conversely, water load decreased plasma osmolality and ϩ cardial endothelial cells.19 plasma Na and AVP concentrations. The decrease in plasma We also observed that in conditions in which water intake was osmolality was accompanied by a rapid increase (within 30 unrestricted and plasma osmolality remained stable within a nar- min) in plasma apelin concentration to a plateau level. Even row range, the plasma apelin concentration remained stable with though the hypo-osmotic stimulus was modest and not sus- Ϫ Ϫ a median within-subject variability of 18.6% (13.3 to 23.6%). It tained ( 4.0 mOsm/kg H2O; 95% confidence interval 7.0 to Ϫ was of the same magnitude as that for plasma AVP concentration. 1.0 mOsm/kg H2O), it was strong enough to maintain the The low within-subject variability contrasting with the high be- plasma apelin concentration at a plateau for 210 min (73.9% tween-subject variability in plasma apelin concentrations may [23.4 to 74.5%]), contrasting with the opposite and modest suggest some genetic influence in the regulation of apelin secre- decrease in plasma AVP concentration (Ϫ30.4% [Ϫ45 to tion, as it has been suggested also for AVP secretion.5,39 Ϫ28.6%]). As expected,4,5 the increase in plasma osmolality induced by The opposite changes in plasma apelin concentration after

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Figure 4. Effects of a 2-h hypertonic saline infusion (0.85 M, 0.06 ml/kg per min E) and a 20-ml/kg water load within 30 min (F)on the plasma apelin/AVP ratio. Data are expressed as medians and IQR. The ANOVA for repeated measurements over time on day 1 was significant in each group. Then pair-wise comparisons were tested using the Dunnett adjustment procedure for multiple tests comparing all time points to baseline level at 9:00 a.m. on day 1. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001 versus baseline (9:00 a.m. on day 1). vation is consistent with plasma osmolality being a major physiologic regulator of plasma apelin levels in humans as previously reported in rodents.10,31 We previously showed that intracerebroventricular injection of apelin inhibited AVP release in the bloodstream in water-deprived and lactating rodents.10,30 Conversely, intracerebroventricular infusion of AVP induced apelin accumulation in magnocellular neurose- cretory neurons; this effect is blocked by a V1 receptor antag- onist.31 Moreover, apelin response to dehydration in rats is opposite to that of AVP. Indeed, water deprivation in rats reduces plasma apelin concentrations and induces an intraneu- ronal pile-up of the peptide in magnocellular AVP neurons, whereas it increases plasma AVP concentrations and decreases AVP neuronal stores because AVP is released faster than it is synthesized.10,31 The increased somatodendritic release of AVP during dehydration in rodents optimizes phasic activity of va- sopressinergic neurons, facilitating systemic AVP release,41 whereas concomitant accumulation of apelin in the same neurons (which is thus not systemically and intranuclearly released), prevents the inhibitory action of apelin on AVP neuron activity, thus facilitating systemic AVP release. The opposite regulation of apelin and AVP during water deprivation in rats allows AVP and apelin to act in concert to reinforce the systemic AVP release necessary for a sustained antidiuresis, thus participating in water balance. It is interesting that our Figure 3. Linear correlations between plasma AVP concentration results show that the same osmotic stimulus leads to an oppo- and plasma osmolality (A), between plasma apelin concentration site regulation of plasma AVP and apelin levels in humans. and plasma osmolality (B), and between plasma apelin concen- This suggests that apelin, like AVP, may participate to the tration and plasma AVP concentration (C) during a 2-h hypertonic saline infusion (0.85 M, 0.06 ml/kg per min). maintenance of body fluid homeostasis in humans as in rodents. the infusion-induced increase or water load–induced decrease Finally, the time course evolution of plasma apelin/AVP in plasma osmolality both were much larger than the within- ratio with changes in osmolality provided additional informa- subject variability in plasma apelin concentration. This obser- tion. Indeed, because baseline plasma AVP concentrations

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Figure 5. (A) Time course of observed (F) and predicted (E) plasma apelin concentration during the hypertonic saline infusion. The equation of the regression line between plasma apelin and plasma osmolality between times 0 and 80 min was then used to predict the curve for plasma apelin between time 80 and time 120 min. (B) Plot of the absolute difference between the observed and the predicted plasma apelin values (left axis, छ) and of the relative increase in estimated extracellular fluid volume (right axis, ࡗ). The observed and predicted values diverged once estimated ECFV had increased by 12 to 15%. Data are means Ϯ SEM. were approximately 150 to 200 times lower than plasma apelin concentrations on a molar basis, the apelin/AVP ratio revealed Figure 6. Effects of a 20-ml/kg water load within 30 min on more significant differences during both the hypertonic saline plasma osmolality (A) and plasma AVP (B) and apelin (C) concen- Ϯ infusion (massive decrease from 196 to 48 fmol/fmol) and the trations. Plasma osmolality and apelin are expressed as means water-loading test (massive increase from 144 to 424 fmol/ SEM. Plasma AVP is expressed as median and IQR. The ANOVA fmol). Thus, the apelin/AVP ratio may indicate the osmolar for repeated measurements over time on day 1 was significant for all three parameters. Then pair-wise comparisons were tested state in the organism, with low values indicating a tendency using the Dunnett adjustment procedure for multiple tests com- toward hyperosmolality and high values a tendency toward paring all time points with baseline level at 9:00 a.m. on day 1. hypo-osmolality. Whether the plasma apelin/AVP ratio may *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001 versus baseline (9:00 a.m. on be a useful index rather than studying each hormone alone in day 1). the diagnosis of hyponatremic/hypo-osmolar states remains to be established in further studies.

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Our study represents the first demonstration in humans of The two experiments (group 1 and group 2) were independent and the regulation of plasma apelin concentration by osmotic stim- performed sequentially at two different periods of the year (autumn uli. We also show that this regulation is opposite to that of to winter for group 1 and late spring to summer for group 2). Each AVP, consistent with results obtained in animal models after group was its own control. water deprivation.10,31,42 This suggests that apelin, like AVP, may participate in the maintenance of body fluid homeostasis Laboratory Methods in humans as in rodents. It is still not clear whether the changes Plasma osmolality was measured by cryoscopy (Fiske Mark3 osmom- in the plasma apelin concentration triggered by changes in eter, Norwood, MA). The methods used for collecting blood samples osmotic pressure only reflect changes in neurohypophyseal se- and for quantifying plasma AVP, active renin (immunoradiometric cretion or have direct peripheral and/or intrarenal hemody- assay), and aldosterone (RIA) were as described previously.44,45 For namic effects15–17 or endocrine effects on water diuresis via plasma apelin measurements, blood was sampled into prechilled 12,18 ϫ binding to intrarenal receptors. Apelin receptor mRNA has EDTA-K3 tubes and centrifuged at 1600 g at 4°C for 15 min and been detected in a subpopulation of cortical glomeruli and in stored at Ϫ80°C. cells along the vasa recta in the inner stripe of the outer me- For HPLC analysis, human plasma samples were prepurified on a dulla, a region of the kidney playing a key role in water and Sep-Pak C18 cartridge (Waters Associates, Milford, MA). Bound ma- sodium balance.12,18 terial was eluted with acetonitrile/50%, and the eluate was evaporated It would be of interest to investigate plasma apelin levels in under reduced pressure. The extract was analyzed by reversed-phase parallel with plasma AVP in various pathologic states of euv- HPLC on a 0.46 ϫ 25-cm Vydac 214TP54 C4 column equilibrated olemic and hypervolemic hyponatremia. Analyzing the rela- with a solution of acetonitrile/water/TFA (10.5:89.4:0.1; vol/vol/vol) tionship between apelin concentration, AVP concentration, at a flow rate of 1 ml/min. The concentration of acetonitrile in the and osmolality in plasma may reveal new classifications of the eluting solvent was raised to 24.5% over 90 min. One-minute frac- multiple causes of hypo-osmolar states of impaired urinary tions were collected and assayed for apelin-IR. Synthetic pE13F, dilution or concentration in patients. Finally, the development K17F, and apelin 36 (2 ␮g each), used as reference peptides, were of nonpeptidic agonists of the apelin receptor could be an al- subjected to chromatography under the same conditions as human ternative or complementary therapeutic approach to V2 recep- plasma samples. tor antagonists43 for the treatment of water retention and/or For apelin RIA, plasma samples (0.75 ml) were mixed with 0.05 ml of hyponatremic disorders. 1% BSA and 0.25 ml of 3 M HCl and centrifuged at 1600 ϫ g at 4°C for 10 min. The supernatants were collected and adjusted to pH 6.5 with 12 M NaOH and 2 M Tris-HCl buffer (pH 7.4). The samples were then mixed CONCISE METHODS with 1 ml of 1% trifluoroacetic acid (TFA) supplemented with 0.05%

BSA and loaded onto a Sep-Pak C18 cartridge (Waters Associates) equil- Participants ibrated with 5 ml of 1% TFA. The columns were washed with 3 ml of 1% Ten healthy normotensive white male nonsmoker volunteers aged 18 to TFA, and apelin peptides were eluted with 1.5 ml of 100% acetonitrile. 25 yr (body mass index 23.5 Ϯ 1.9 kg/m2) completed the study after The recovery (mean Ϯ SEM) was 91 Ϯ 3%. The eluates were dried and giving written and informed consent. The protocol was approved by the dissolved in 0.55 ml of RIA buffer. ethics committee “CCPPRB, Paris-Cochin, France,” and the investiga- Immunoreactive apelin concentration was measured by RIA using tion was conducted according to Declaration of Helsinki principles. highly selective rabbit polyclonal antibodies directed against the apelin fragment K17F produced in the laboratory.30 This antibody recog- Study Protocol nizes two epitopes in K17F, one of them being common to pE13F and Participants were instructed to arrive at the Clinical Investigation K17F. We thus used iodinated pE13F as a tracer to make sure to Center at 7:00 a.m. on day Ϫ1 and remained hospitalized for 36 h. measure equivalently K17F and pE13F endogenous levels. When us- Fluid intake throughout day Ϫ1 was unrestricted until 9:00 p.m., and ing iodinated pE13F as a tracer and defining reactivity with pE13F as participants were given light meals at 9:30 a.m., 12:00 p.m., and 8:00 100%, our antibodies cross-react 214% with K17F, 100% with pE13F, p.m. (NaCl approximately 150 mmol/d). From 9:00 p.m. until 9:00 and 100% with apelin 36. Reactivity decreased with loss of amino a.m. on day 1, participants refrained from water and food intake. At acids from the N-terminal part of K17F and was negligible with C- 9:00 a.m. on day 1, after a 12-h overnight fast and water restriction, terminally truncated fragments of K17F and various other bioactive five participants received an infusion of a 2-h 0.85-M saline solution peptides, including angiotensin II, angiotensin III, neuropeptide Y, (5%) at the rate of 0.06 ml/kg per min to increase plasma osmolality and AVP. Plasma samples (0.1 ml) were mixed with [125I]pE13F (group 1). The five remaining participants ingested 20 ml/kg body wt (15,000 cpm) and K17F antiserum (1:10,000) to give a total volume of water within 30 min to decrease plasma osmolality (group 2). In the 0.2 ml and incubated at 4°C overnight. Then, the antigen–antibody two groups, blood was sampled at various times on days Ϫ1 and 1 for complex was immunoprecipitated and quantified as described previ- plasma osmolality, Naϩ, apelin, AVP, active renin, aldosterone, he- ously.10 The detection limit was 6 fmol/ml, and the quantification matocrit, and total protein determinations with the participants being limit was 12 fmol/ml. The within- and between-assay coefficients of semirecumbent for 1 h before each blood sampling. Urine was col- variation were 3 and 5%, respectively. Serial dilutions of human lected before and during the tests for determination of electrolytes. plasma samples gradually inhibited binding of [125I]pE13F to the an-

1022 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1015–1024, 2008 www.jasn.org CLINICAL RESEARCH tiserum, and inhibition curves paralleled that of pE13F used as a stan- Kitada C, Nishizawa N, Murosaki S, Kurokawa T, Onda H, Tatemoto K, dard (data not shown). Fujino M: Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim Biophys Acta 1452: 25–35, 1999 Statistical Analyses 9. Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, Osmond Data are expressed as medians with IQR for variables known to be DH, George SR, O’Dowd BF: Characterization of apelin, the ligand for non-normally distributed and for all relative variations from baseline. the APJ receptor. J Neurochem 74: 34–41, 2000 For all other measurements, data are expressed as means Ϯ SD. Data 10. De Mota N, Reaux-Le Goazigo A, El Messari S, Chartrel N, Roesch D, Dujardin C, Kordon C, Vaudry H, Moos F, Llorens-Cortes C: Apelin, a were analyzed using ANOVA for repeated measurements over time potent diuretic neuropeptide counteracting vasopressin actions with a modeling covariance structure within subjects. When the through inhibition of vasopressin neuron activity and vasopressin re- global time effect was significant, pair-wise comparisons were tested lease. Proc Natl Acad Sci U S A 101: 10464–10469, 2004 using the Dunnett adjustment procedure for multiple tests. Correla- 11. Kawamata Y, Habata Y, Fukusumi S, Hosoya M, Fujii R, Hinuma S, tions between variables were estimated using analysis of covariance Nishizawa N, Kitada C, Onda H, Nishimura O, Fujino M: Molecular Ͻ properties of apelin: Tissue distribution and receptor binding. Biochim after withdrawal of the subject effect. P 0.05 was considered to be Biophys Acta 1538: 162–171, 2001 significant. SAS statistical software version 8.2 (Cary, NC) was used 12. Medhurst AD, Jennings CA, Robbins MJ, Davis RP, Ellis C, Winborn for statistical analyses. KY, Lawrie KW, Hervieu G, Riley G, Bolaky JE, Herrity NC, Murdock P, Darker JG: Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin. J Neuro- chem 84: 1162–1172, 2003 ACKNOWLEDGMENTS 13. Zhou N, Zhang X, Fan X, Argyris E, Fang J, Acheampong E, DuBois GC, Pomerantz RJ: The N-terminal domain of APJ, a CNS-based This work was supported by a joint grant from the Assistance Pub- coreceptor for HIV-1, is essential for its receptor function and coreceptor activity. Virology 317: 84–94, 2003 lique des Hoˆpitaux de Paris (CRC 04128 and Contrat d’Interface 14. De Mota N, Lenkei Z, Llorens-Cortes C: Cloning, pharmacological P-1811-2004), Association Robert Debre´, Naturalia & Biologia, and characterization and brain distribution of the rat apelin receptor. Neu- the National Research Agency program (ANR-05-PCOD-001-01). roendocrinology 72: 400–407, 2000 We thank the nursing staff of the Clinical Investigation Center 15. Masri B, Knibiehler B, Audigier Y: Apelin signalling: A promising who ran the protocol and Dr. Laure Marelle, who recruited the pathway from cloning to pharmacology. Cell Signal 17: 415–426, 2005 healthy participants. The help of Christiane Dollin, who performed 16. Kleinz MJ, Davenport AP: Emerging roles of apelin in biology and the renin, the aldosterone, and the AVP determinations, is much ap- medicine. Pharmacol Ther 107: 198–211, 2005 preciated. 17. Lee DK, George SR, O’Dowd BF: Unravelling the roles of the apelin system: Prospective therapeutic applications in heart failure and obesity. Trends Pharmacol Sci 27: 190–194, 2006 18. 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