J Am Soc Nephrol 11: 1293–1302, 2000 Nitric Oxide Synthesis Inhibition Does Not Impair Water Immersion-Induced Renal Vasodilation in Humans LIOE-TING DIJKHORST-OEI, PETER BOER, TON J. RABELINK, and HEIN A. KOOMANS Department of Nephrology and Hypertension, University Hospital Utrecht, The Netherlands. Abstract. Nitric oxide (NO) is tonically released in the kidney increased sodium excretion (UNaV) from 110 Ϯ 27 to 195 Ϯ to maintain renal perfusion and adequate sodium and water 29 ␮mol/min and flow (UV) from 14.4 Ϯ 1.4 to 15.8 Ϯ 1.4 clearance. Little is known about the role of NO in the renal ml/min. L-NMMA caused profound and sustained increases in adaptation to an acute volume challenge. This is important for MAP and RVR and decreases in UNaV and UV. HOI super- our understanding of pathophysiologic conditions associated imposed on L-NMMA infusion decreased the elevated MAP with impaired NO activity. This study examined the effects of from 93 Ϯ 4to83Ϯ 2 mmHg and RVR from 111 Ϯ 9to95Ϯ NO synthesis inhibition on neurohumoral, renal hemodynamic, 7 mmHg ⅐ min/L, and increased UNaV from 41 Ϯ 8to95Ϯ and excretory responses to head-out immersion (HOI). Seven 15 ␮mol/min and UV from 10.0 Ϯ 1.1 to 12.7 Ϯ 1.4 ml/min. healthy men underwent four 7-h clearance studies. One study The relative changes were not significantly different from the served as a time control study (placebo infusion), and in one effects of HOI without L-NMMA infusion. HOI decreased G study N -monomethyl-L-arginine (L-NMMA; 3 mg/kg priming plasma renin activity and aldosterone and increased plasma dose ϩ 3 mg/kg per h) was infused during hours 2 to 5. In a atrial natriuretic peptide and urinary cGMP. L-NMMA de- third and fourth clearance study, HOI was applied from hours creased urinary cGMP, but did not affect the plasma hormones 15 3 to 5, during infusion of either placebo or L-NMMA. To assess or the changes induced by HOI. L-NMMA decreased the [ N]- 15 the degree of NO synthesis inhibition, the effect of L-NMMA arginine-to-[ N]-citrulline conversion rate to one-third of on [15N]-arginine-to-[15N]-citrulline conversion rate was stud- baseline. The results indicate that in a state of NO deficiency in ied in four others. HOI decreased mean arterial pressure humans, the kidney can still respond to an acute volume (MAP) from 87 Ϯ 3to76Ϯ 2 mmHg and renal vascular challenge with vasorelaxation, diuresis, and natriuresis. resistance (RVR) from 82 Ϯ 6to70Ϯ 7 mmHg ⅐ min/L, and Head-out immersion (HOI) is a model of acute central blood of NO synthesis, for instance by bradykinin, induces marked volume expansion. The renal response to this maneuver con- vasodilation natriuresis and diuresis (5). That NO plays a role sists of renal vasorelaxation and diuresis and natriuresis (1). in the physiologic response to HOI is therefore conceivable. Thus, HOI can be used to study the factors that play a role in However, because many other mediators are involved, the role acute modulation of renal hemodynamics and excretory func- of NO could be purely a modulating one rather than a medi- tion. Among these are suppression of the renin-angiotensin ating one. system and sympathetic tone, and stimulation of atrial natri- Apart from being relevant for normal physiology, this issue uretic peptide (ANP) and renal vasodilatory prostaglandins (1). is relevant for our understanding of essential hypertension. Whether intact nitric oxide (NO) availability is essential for This condition has been associated with impaired availability a normal renal response to HOI has not been investigated. of NO (6–8). Patients with essential hypertension display an Blockade of NO synthesis in animals (2) and humans (3,4) impaired renal vasodilatory response to L-arginine infusion (9). induces strong renal vasoconstriction, accompanied by sodium This is particularly the case in patients with salt-sensitive and water retention, which underscores the importance of NO hypertension (10), a condition that may be specifically related in keeping the kidney tonically in a vasodilated state and to impaired NO activity (11,12). Nonetheless, the renal vaso- permitting adequate sodium and water excretion. Stimulation dilatory and natriuretic response to an acute volume challenge is normal or paradoxically increased (13–16). We therefore studied the acute renal responses to HOI in Received September 17, 1998. Accepted October 9, 1999. healthy humans with and without NO synthesis inhibition G Correspondence to Dr. Hein A. Koomans, Department of Nephrology and induced by infusion of N -monomethyl-L-arginine (L- Hypertension, University Hospital Utrecht, Room F03.226, P.O. Box 85500, NMMA). This NO synthesis inhibitor was infused for several 3508 GA Utrecht, The Netherlands. Phone: ϩ31 30 2507329; Fax: ϩ31 30 2543492; E-mail: [email protected] hours to induce steady-state conditions. The magnitude of NO synthesis inhibition was assessed from the effect of L-NMMA 1046-6673/1106-1293 15 15 Journal of the American Society of Nephrology on the [ N]-arginine-to-[ N]-citrulline conversion rate. De- Copyright © 2000 by the American Society of Nephrology tails of this technique were published recently (17). 1294 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 1293–1302, 2000 Materials and Methods Clearances were calculated according to standard formula. Mean Water Immersion Experiments arterial pressure (MAP) was calculated as the sum of one-third of Studies were carried out in seven healthy men (age range, 20 to 25 systolic pressure and two-thirds of diastolic pressure. Renal blood yr). Their health status was assessed by medical history, physical flow was calculated by dividing ERPF by (1-packed cell volume). MAP was divided by renal blood flow to estimate renal vascular examination, and routine laboratory investigation. All participants resistance. The changes induced by HOI were corrected for the gave written informed consent after extensive explanation of the changes found during the control studies. For this purpose, the ratios protocol. The study protocol was approved by the University Hospital of the individual values during the HOI study and the corresponding Ethical Committee for Studies in Humans. control study were calculated for each urine collection period. Each subject underwent two clearance experiments while remain- ing seated in air (air temperature 26.0 Ϯ 1.0°C), and two clearance experiments while undergoing HOI for 3 h. In either two conditions, [15N]-Arginine-to-[15N]-Citrulline Conversion Rate one experiment was performed during L-NMMA infusion to inhibit Experiments NO synthesis and the other during placebo (vehicle) infusion. Infusion As a measure of whole-body NO synthesis before and during the of either L-NMMA or placebo was started after completion of the 1-h NO synthase (NOS) blockade, the [15N]-arginine-to-[15N]-citrulline baseline collections. L-NMMA (Institut fu¨r Pharmazie, Universita¨t conversion rate was determined in four healthy volunteers (3 male, 1 Leipzig, Germany) was dissolved in normal saline (5 mg/ml) and female, age range 22 to 28 yr) at baseline and until 2 h after the start infused as a priming dose of 3 mg/kg body wt, followed by mainte- of L-NMMA infusion, using the same protocol as in the clearance nance infusion of 3 mg/kg per h during 4 h, from hours 2 to 5. This studies (3 mg/kg priming dose, followed by 3 mg/kg per h). The scheme was chosen to achieve a period of steady state, such as is protocol was approved by the University Hospital Ethical Committee desired for studies using clearance techniques. The average volume for Studies in Humans, and subjects provided written informed con- infused was 220 ml over 4 h. In the two HOI studies, after1hof sent after explanation of the protocol. Ϯ infusion subjects were immersed in thermoneutral water (35.5 Two hours before L-NMMA infusion was started, two blood sam- 0.5°C) up to the sternoclavicular notch. Immersion was thus applied ples were obtained for determination of (natural) background isotope from hour 3 to hour 5, with only brief interruptions for urine collec- ratios. Then subjects received a priming dose of 12 ␮mol L-[gua- tion, and ended at the same time as the infusion period. After cessation 15 Ͼ nidino- N2O]-arginine (purity 98% atom percent excess (APE); of the infusion period, recovery was observed for 2 more hours. Mass Trace, Woburn, MA) per kilogram body weight in 2 min, The 7-h clearance studies were carried out on separate days at least followed by a constant infusion of 11.2 ␮mol/kg per h. After 1 h, three 3 d apart. The order of the studies was randomized. The studies were blood samples for measurement of steady-state baseline enrichments performed after5dofadiet containing 200 mmol sodium. Adherence were collected at 30-min intervals. Subsequently, L-NMMA adminis- to the diet was monitored by 24-h urine collections on the day before tration was started and blood sampling at 30-min intervals was con- each clearance experiment. tinued for another 2 h. On the eve of each clearance study, 400 mg of lithium carbonate Arginine and citrulline 15N enrichment in plasma was determined was ingested at 10 p.m. The experiments were started by subjects by electrospray ionization mass spectrography with a VG Platform receiving an oral water load of 25 ml/kg between 7:30 a.m. and single quadrupole instrument after separation using a Pharmacia 8.30 a.m. Additional water-matching urine output was supplied Smart System, equipped with a 4 ϫ 250 mm Sephasil C18 column, a throughout the clearance study.