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Am J Physiol Renal Physiol 274: F914-F923, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 5, F914-F923, May 1998

Dietary sodium affects systemic and renal hemodynamic response to NO inhibition in healthy humans

J. N. Bech, C. B. Nielsen, P. Ivarsen, K. T. Jensen, and E. B. Pedersen

Research Laboratory of Nephrology and Hypertension, Aarhus Amtssygehus, University Hospital in Aarhus, 8000 Aarhus C, Denmark

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal studies have indicated that increased nitric oxide (NO) synthesis plays a significant role in the renal adaptation to increased sodium intake. To investigate the role of NO during increased sodium intake in humans, we studied the effect of acute, systemic injection of NG-monomethyl-L-arginine (L-NMMA) on renal hemodynamics [glomerular filtration rate and renal plasma flow (GFR and RPF, respectively)], urinary sodium excretion (FENa), systemic hemodynamics [mean arterial blood pressure and heart rate (MAP and HR)], and plasma levels of several vasoactive hormones in 12 healthy subjects during high (250 mmol/day) and low (77 mmol/day) sodium intake in a crossover design. The sodium diets were administered for 5 days before the L-NMMA treatments, in randomized order, with a washout period of 9 days between each diet and L-NMMA treatment. GFR and RPF were measured using the renal clearance of 51Cr-labeled EDTA and 125I-labeled hippuran by the constant infusion technique in clearance periods of 30-min duration. Two baseline periods were obtained, after which L-NMMA was given (3 mg/kg over 10 min), and the effect of treatment was followed over the next five clearance periods. During high sodium intake, L-NMMA induced a more pronounced relative decrease in RPF (P = 0.0417, ANOVA), a more pronounced relative decrease in FENa (P = 0.0032, ANOVA), and a more pronounced relative increase in MAP (P = 0.0231, ANOVA). During low sodium intake, the effect of L-NMMA on FENa was abolished. During low sodium intake, L-NMMA induced a sustained drop in plasma renin (31 ± 5 vs. 25 ± 5 µU/ml, P < 0.001), which was not seen during high sodium intake. The data indicate that increased production of NO is an important part of the adaptation to increased dietary sodium intake in healthy humans, with respect to renal hemodynamics, sodium excretion, and the secretion of renin.

nitric oxide; renal plasma flow; glomerular filtration rate; dietary sodium

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A LARGE BODY OF recent experimental evidence indicates that the gaseous molecule nitric oxide (NO) is an important regulator of systemic blood pressure, renal hemodynamics, and renal sodium and water excretion during basal conditions (19, 28). Systemic or intrarenal administration of unselective inhibitors of nitric oxide synthase (NOS) such as NG-monomethyl-L-arginine (L-NMMA) or Nomega -nitro-L-arginine methyl ester in animals or humans leads to increased systemic blood pressure, renal vasoconstriction, and decreased sodium and water excretion (4, 7, 19, 35). Additional animal evidence points to NO as a critical modulator of sodium excretion and long-term blood pressure regulation. Animals treated chronically with NOS inhibitors develop hypertension, with a resetting of the pressure-natriuresis relation toward higher blood pressures (24). Animals treated with lower (subpressor) doses of NOS inhibitor develop hypertension only after sodium loading (29). Moreover, several animal studies have provided evidence of an increased impact of NO during sodium loading on systemic blood pressure and renal hemodynamics (7, 30, 35).

Recently, we investigated the effects of systemic L-NMMA treatment in healthy, sodium-replete humans, and the results were in agreement with most of the findings in animal studies (4). However, at present, there are no human data on the significance of NO during the renal adaptation to increased sodium intake in terms of renal hemodynamics and urinary sodium excretion (UNaV). In addition, there are no human data describing the effects of NO on water excretion and the plasma levels of vasoactive hormones during different levels of sodium intake. Interactions among NO and the secretion and/or effect of renin, vasopressin, and other hormones have only been described in animals.

To provide human data on these issues, we decided to study the effects of acute, systemic NO synthesis inhibition with L-NMMA in healthy subjects, adapted to either high (HS) or low (LS) sodium intake, on renal hemodynamics, UNaV, water excretion, and plasma levels of several hormones of interest in the regulation of blood pressure, sodium balance, and renal hemodynamics. As changes in plasma levels of L-arginine (L-Arg, a NO substrate) have been implicated as a regulatory mechanism of NO synthesis during LS intake, we also measured baseline values of L-Arg during both HS and LS intake.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Subjects and Design

The study was approved by the local ethics committee and The Danish National Board of Health. Informed consent was obtained from all subjects. The study was performed as a randomized crossover investigation. Twelve healthy subjects were included in the study (age, 23.8 ± 0.5 yr; males, n = 6; females, n = 6). The subjects were studied on 2 study days during acute administration of L-NMMA. Before the 1st study day, the subjects were randomized to a diet treatment (see below) with either LS or HS content. The diet treatment was followed for 5 days prior to the 1st study day. After the 1st study day and after a washout period of 9 days, the subjects entered another diet period of 5 days with the other sodium content. The 2nd-day study was then performed. The following inclusion criteria were used. Both men and women, ages <40 yr, had no evidence or history of disease of the heart, liver, kidneys, or endocrine organs, no history of alcohol or drug abuse, and no current medical treatment. Prior to the study, all subjects had a clinical examination, blood pressure measurement, electrocardiogram, and laboratory screening, including hematology, S-electrolytes, S-creatinine, B-glucose, S-cholesterol, S-bilirubin, alkaline phosphatase, and prothrombine time. Urine was analyzed by dipstick. The results of these tests were required to be completely normal before inclusion.

Sodium Diet

The diet treatment was performed on an ambulatory basis with self-prepared meals. Adherence to the diets was controlled by obtaining written dietary histories, 24-h UNaV, and 24-h urinary urea excretion on the last day of each diet treatment. LS diet contained 77 mmol/day sodium (9,500 kJ/day) and 72 g/day of protein. HS diet contained similar amounts of energy and protein and a sodium content of 250 mmol/day.

L-NMMA Treatment and Clearance Examinations

The subjects were instructed to fast overnight before each of the study days. On the study days, an oral water load of 200 ml tap water every 30 min was started at 0700. Two in-dwelling catheters for blood sampling and administration of tracers and study drugs were placed in forearm veins, one in each arm. Urine was collected by voiding in the standing or sitting position, and the subjects were otherwise kept in the supine position during the experiment. At 0830, a priming dose of 51Cr-labeled EDTA and 125I-labeled hippuran for the measurement of glomerular filtration rate (GFR) and renal plasma flow (RPF) was given followed by sustain infusions of both. The study was divided into seven 30-min clearance periods. Two baseline periods were obtained from 1000 to 1100. At 1100, a bolus injection of L-NMMA (3 mg/kg) dissolved in 10 ml of saline was given over 10 min. The study continued with five clearance periods to evaluate the effects of treatment. Blood samples were drawn every 30 min, i.e., before the first clearance period and at the end of each period and were analyzed for tracers, osmolality, and electrolytes. In addition, analysis of the plasma levels of guanosine 3',5'-cyclic monophosphate (PcGMP), renin, aldosterone (Aldo), angiotensin II (ANG II), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and endothelin-1 (ET-1) were performed from samples withdrawn at 1100 (baseline) and from samples withdrawn at 1200 (60 min after injection), at 1300 (120 min after injection), and at 1400 (180 min after injection). Plasma levels of L-Arg and citrulline (Cit) were obtained from samples withdrawn at 1000. Urinary collections were analyzed for tracers, electrolytes, osmolality, and cGMP.

GFR and RPF were measured by the constant infusion clearance technique using 51Cr-EDTA and 125I-hippuran as reference substances. A priming dose ensured a serum activity of 200-600 cpm/ml for 51Cr-EDTA and 800-1,600 cpm/ml for 125I-hippuran. Serum activity was kept stable by constant intravenous infusion with an infusion pump (Vial Medical).

Blood pressures were determined by a semiautomatic device (Takeda) calibrated against a random-zero sphygmomanometer (Hawksley, Lancing, UK).

Biochemical Measurements

cGMP in plasma and urine was measured using a radioimmunoassay kit (Amersham). Ethanol was used for extraction from plasma. The coefficients of variation were 9% (interassay) and 6% (intra-assay). Minimal detection level was 2.5 pmol/l plasma.

ANG II in plasma was measured by radioimmunoassay by a modification of the method described by Kappelgaard et al. (18). Radioimmunoassay was performed after previous extraction from plasma by Sep-Pak C18 cartridges (Water Associates, Milford, MA). The antibody was obtained from the Dept. of Clinical Physiology, Glostrup Hospital (Glostrup, Denmark). Minimal detection level was 2 pmol/l plasma. The coefficients of variation were 12% (interassay) and 8% (intra-assay).

Aldo in plasma was measured by a slight modification of a previously described method (26). With a rabbit anti-Aldo antibody (International CIS), radioimmunoassay was performed on a residue from plasma prepared by extraction with dichloromethane and purification on silica gel columns. Minimum detection level was 42 pmol/l. The coefficients of variation were 13% (interassay) and 9% (intra-assay).

ANP in plasma was determined by radioimmunoassay, as previously described (34). ANP was extracted from plasma by means of Sep-Pak C18 cartridges. For radioimmunoassay, rabbit anti-ANP antibody was obtained from the Dept. of Clinical Chemistry, Bispebjerg Hospital (Copenhagen, Denmark). The minimum detection level was 0.5 pmol/l plasma. The coefficients of variation were 12% (interassay) and 10% (intra-assay).

BNP in plasma was measured by a radioimmunoassay developed in our laboratory (17). Immunoreactive BNP was extracted from plasma by use of Sep-Pak C18 cartridges eluted by 80% ethanol in 4% acetic acid. Radioimmunassay was performed using a rabbit anti-BNP antibody developed in our laboratory. There was no cross-reactivity with ANP. The minimum detection level was 0.55 pmol/l. The coefficients of variation were 6% (intra-assay) and 11% (interassay).

Renin in plasma was measured using a two-site immunoradiometric assay (Nichols). The coefficients of variation were 2.5% (intra-assay) and 9.9% (interassay). Minimal detection level was 1.4 µU/ml.

ET-1 in plasma was measured, using a solid-phase ELISA, containing human ET-1 and antibodies raised against synthetic human ET-1 (R & D Systems). The coefficients of variation were 4.5% (intra-assay) and 5.5% (interassay). Minimal detection level was 0.25 pg/ml.

Plasma concentrations of L-ARG and Cit were determined using HPLC, with a slight modification of the method described by Graser et al. (12). Precipitation of protein was done with ice-cold 4% sulfosalicylic acid, and samples were stored at -80°C until analysis. Precolumn derivatization was performed with O-phthaldialdehyde. The coefficients of variation for determination of L-ARG and Cit were 3.5 and 5.7%.

Plasma and urinary concentrations of sodium and potassium were measured by routine methods at the Dept. of Clinical Chemistry, Skejby Hospital. Plasma and urinary osmolality was measured by freezing-point depression (Advanced Cryomatic Osmometer, model 3C2).

Study Drug

Pharmaceutical-grade L-NMMA was obtained from Clinalfa.

Calculations and Statistics

All clearance calculations are based on standard formulae, using the mean value of the two plasma levels before and after each clearance period. Renal vascular resistance (RVR) was calculated as mean arterial blood pressure (MAP)/RPF. All results are given as means ± SE. Dietary sodium treatment significantly affected baseline values of some of the study parameters. We therefore compared relative changes from baseline when comparing the effects of LS vs. HS treatment. Within-group comparisons were performed by analysis of variance with repeated measures design and paired Student's t-tests with Bonferroni correction as post hoc tests. Comparisons between HS and LS treatment were performed using paired Student's t-test (baseline comparisons) or two-way repeated-measures analysis of variance with time and sodium treatment as factors. All variables were normally or log-normally distributed. P < 0.05 was considered the limit of significance.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of Sodium Diet

Sodium diet elicited profound changes in several effect parameters at baseline. The measurements of 24-h sodium excretion (Table 1) indicated a satisfactory overall adherence to the diet. HS balance was accompanied by a gain in body weight and increased levels of plasma sodium and plasma osmolality (Table 1). There were no changes in MAP or heart rate (HR) (Table 1). In addition, during HS, GFR and RPF increased significantly and RVR decreased significantly (Table 2). As expected, LS balance was accompanied by a significant activation of the components of the renin-angiotensin-aldosterone axis (renin, Aldo, ANG II; Table 4) and a significant relative suppression of the natriuretic peptides (ANP and BNP, Table 4). Indices of NO production, such as plasma (PcGMP) and urinary (UcGMP) cGMP (Table 4) and Cit (Table 1) were all significantly increased during HS balance. The diet did not influence plasma levels of L-Arg, the substrate of NO synthesis (Table 1).

                              
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  		Table 1.   Clinical and laboratory characteristics of 12 healthy subjects after low sodium and high sodium intake

                              
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  		Table 2.   Effects of injection of L-NMMA (3 mg/kg) on GFR, RPF, RVR, and FF in 12 healthy individuals during HS and LS intake

Effects of L-NMMA

Blood pressure and heart rate. Figure 1 shows relative changes in MAP and HR after L-NMMA treatment during LS and HS treatment. During HS diet, L-NMMA elicited a maximum increase in MAP 10 min after injection (81 ± 2 vs. 90 ± 2 mmHg, P < 0.0001) and also during LS diet (80 ± 2 vs. 87 ± 2 mmHg, P = 0.0030). The increase in MAP was more pronounced and of longer duration during HS, where the increase was significant up to 40 min after injection, as opposed to during LS, where the increase was significant only after 10 min (Fig. 1). In terms of relative changes in MAP, the maximum increase in MAP was almost twofold during HS, compared with LS (Fig. 1, P = 0.0231). During both HS and LS, L-NMMA induced an immediate reduction in HR after 10 min (HR-HS: 48 ± 2 vs. 56 ± 3 beats/min, P < 0.0001; HR-LS: 51 ± 2 vs. 57 ± 2 beats/min, P = 0.0006) that normalized within 30 min. Sodium diet did not influence the relative decrease in HR significantly (Fig. 1, P = 0.7545).


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  		Fig. 1.   Relative change (percent) in mean arterial blood pressure (MAP, top) and heart rate (bottom) during high (HS, black-square) and low (LS, square ) sodium intake after treatment with NG-monomethyl-L-arginine (L-NMMA) (3 mg/kg) in 12 healthy subjects. Data are means ± SE. * P < 0.05 vs. baseline with ANOVA.

Renal hemodynamics. The absolute values of RPF, RVR, GFR, and filtration fraction (FF) before and after L-NMMA are shown in Table 2. The relative changes in GFR and RPF are depicted in Fig. 2. During HS, L-NMMA induced a drop in GFR which was maximal after 150 min (-10.0 ± 1.6% vs. baseline, P = 0.0001) and a decrease in RPF, which was maximal after 30 min (-18.4 ± 2.2% vs. baseline, P < 0.0001). The drop in RPF lasted throughout the experiment. During LS, L-NMMA similarly induced a drop in GFR with a maximum after 30 min (-9.5 ± 1.3% vs. baseline, P < 0.0001) and a decrease in RPF, which was maximal after 30 min (-19.9 ± 1.9% vs. baseline, P < 0.0001). The relative changes in GFR were not significantly different during LS and HS (Fig. 2, P = 0.4321). The relative decrease in RPF was significantly greater during HS in terms of duration of the response (Fig. 2, P = 0.0417). RVR increased significantly with a maximum after 30 min during both HS (+32.8 ± 4.9% vs. baseline, P < 0.0001) and LS (+30.5 ± 3.0% vs. baseline, P < 0.0001). The relative increase in RVR was significantly more pronounced during HS during the last four periods of the experiments (Fig. 2, P = 0.0408, ANOVA). FF increased after 30 min to a similar extent after L-NMMA in both groups (FF-HS: +13.0 ± 2.0% vs. baseline, P < 0.0001; FF-LS: +13.3 ± 1.5% vs. baseline, P < 0.0001). Over the course of the experiment, there was no significant difference among the relative changes in FF (data not shown, P = 0.4410).


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  		Fig. 2.   Relative change (percent) in renal plasma flow (RPF, top), glomerular filtration rate (GFR, middle), and renal vascular resistance (RVR, bottom) during HS and LS intake after treatment with L-NMMA (3 mg/kg) in 12 healthy subjects. Symbols and abbreviations in Figs. 2-4 are as in Fig. 1. Data are means ± SE. * P < 0.05 vs. baseline with ANOVA.

Urine flow rate, urinary osmolality, sodium excretion, and free water clearance. Absolute values of urinary flow rate (V), UNaV, fractional sodium excretion (FENa), urinary osmolality (Uosm), and free water clearance (CH2O) before and after L-NMMA treatment are given in Table 3. The relative values of FENa, Uosm, and CH2O are depicted in Fig. 3. During HS, L-NMMA treatment induced a significant and sustained decrease in UNaV and FENa with a maximum 60 min after injection (UNaV-HS: -23.0 ± 7.7% vs. baseline, P = 0.0010; FENa-HS: -17.5 ± 5.3% vs. baseline, P = 0.0019). During LS, L-NMMA treatment did not affect UNaV or FENa. The relative changes in UNaV and FENa were significantly different between HS and LS [UNaV (data not shown), P = 0.0040; FENa (Fig. 3), P = 0.0032, respectively].

                              
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  		Table 3.   Effects of injection of L-NMMA (3 mg/kg) on V, UNaV, FENa, Uosm, and CH2O in 12 healthy individuals during HS and LS intake


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  		Fig. 3.   Relative change (percent) in fractional excretion of sodium (FENa, top), free water clearance (CH2O, middle), and urinary osmolality (Uosm, bottom) during HS and LS intake after treatment with L-NMMA (3 mg/kg) in 12 healthy subjects. Data are means ± SE. * P < 0.05 vs. baseline with ANOVA.

L-NMMA treatment was accompanied by a significant decrease in V on both study days, lasting throughout the experiment with a maximum decrease after 30 min (V-HS: -41.6 ± 5.5% vs. baseline, P < 0.0001; V-LS: -47.4 ± 6.1% vs. baseline, P = 0.0001). The relative changes in V from baseline tended to be enhanced during LS; however, these were not significant (data not shown, P = 0.0632).

The decrease in V after L-NMMA was accompanied by an increase in Uosm, with a maximum after 30 min and lasting for 60 min after treatment but only during LS (Uosm-HS: +20.6 ± 6.4% vs. baseline; P, not significant; Uosm-LS: +75.4 ± 18.4% vs. baseline, P = 0.0075). Thus the relative increase in Uosm after L-NMMA was greatly enhanced during LS compared with HS (Fig. 3, P = 0.0006).

CH2O decreased significantly after L-NMMA treatment during both HS and LS with a maximum after 30 min (CH2O-HS: -54.5.1 ± 8.8% vs. baseline, P = 0.0002; CH2O-LS: -67.6 ± 12.0% vs. baseline, P = 0.0001). However, during LS, the decrease in CH2O was more pronounced and sustained throughout the experiment, and the difference between the relative changes in the two groups was significant (Fig. 3, P = 0.0261).

Hormones. Plasma levels of vasoactive hormones and UcGMP are shown in Table 4, before and after L-NMMA injection during HS and LS. During LS, L-NMMA induced a sustained drop in plasma renin that lasted throughout the experiment and after the normalization of systemic blood pressure (Fig. 4). This was not seen during LS. Plasma levels of ANP decreased significantly on both study days 120 min after L-NMMA. Levels of ANG II, Aldo, BNP, and ET-1 were not affected by L-NMMA treatment. PcGMP and UcGMP decreased promptly and significantly after L-NMMA in both groups. The relative decrease in UcGMP was significantly more steep during HS (Fig. 4, P = 0.0060).

                              
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  		Table 4.   Effects of injection of L-NMMA (3 mg/kg) on plasma levels of vasoactive hormones and UcGMP in 12 healthy individuals during HS and LS intake


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  		Fig. 4.   Relative change (percent) in urinary excretion of cGMP (UcGMP, top) and plasma renin (bottom) during HS and LS intake after treatment with L-NMMA (3 mg/kg) in 12 healthy subjects. Data are means ± SE. * P < 0.05 vs. baseline with ANOVA.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study describes the effects of acute, systemic NO synthesis inhibition on renal hemodynamics, sodium and water excretion, systemic blood pressure, and the plasma levels of vasoactive hormones during HS and LS intake in healthy humans. We were able to demonstrate a more pronounced relative increase in MAP and a more pronounced relative decrease in RPF, UNaV, and FENa during HS intake as a result of NO synthesis inhibition. On the other hand, the decrease in CH2O was more pronounced during LS intake.

Increased NO activity during HS was indicated by increased plasma levels and urinary excretion of cGMP and increased plasma levels of Cit, which is a split product of NO synthesis. Moreover, there was a more pronounced effect of L-NMMA on UcGMP during HS. However, it should be emphasized that these parameters are only indirect measures of NO activity. cGMP levels reflect the combined activity of both NO and the natriuretic peptides, of which we measured ANP and BNP. Clearly, the increased plasma levels and urinary excretion of cGMP as a result of HS intake could be attributed partly to the enhanced activity of the natriuretic peptides seen in this study. The drop in PcGMP and UcGMP after L-NMMA occurred promptly and is most likely the result of NO synthesis inhibition. We did measure a drop in ANP, which occurred significantly later in the experiment. This drop in ANP could possibly explain part of the drop in cGMP levels.

We found that the dietary intervention did not change plasma levels of L-Arg, the substrate of NO synthesis, suggesting that substrate availability was not influenced by the diet. Studies by Deng et al. (8) have suggested that a decreased availability of L-Arg may downregulate NO synthesis during LS intake. However, the present study does not exclude the possibility that cellular transport of L-Arg is changed as a result of LS balance.

The increase in MAP after L-NMMA was clearly enhanced during HS, suggesting an increased dependence of the vasodilatory action of NO during sodium loading in the systemic circulation. In theory, this response could also be explained by an increased unopposed vasoconstrictory tone during HS. However, this notion seems hard to accept in face of the suppression of the renin-angiotensin system and unchanged plasma levels of ET-1 occurring during HS in this study. In addition, acute systemic hypertension still develops in binephrectomized rats after systemic NO inhibition (9). An increased sensitivity of the forearm vasculature in humans during HS intake to the NO donor nitroprusside, but not metacholine, has been reported recently (31). Metacholine is thought to act via endothelial cholinergic receptors to stimulate vascular NO synthesis. These results indicate that increased sodium intake leads to an increased vascular sensitivity to the actions of NO. It is still possible that vascular NOS expression is increased during HS intake, but to our knowledge, no studies have dealt directly with this issue. The hypertensive effect of systemic NO inhibition is probably the result of several mechanisms other than the inhibition of systemic vascular NOS. A growing number of studies have identified NO-sensitive potassium channels that account for a part of the vasodilatory action of NO (5). The expression and relative importance of these potassium channels during different levels of sodium intake is not known. In addition, studies have shown that, in the rat, systemic hypertension could be elicited by intracerebroventricular administration of NOS inhibitor (9). These findings indicate a profound importance of centrally acting NO in the regulation of systemic blood pressure. The relative influence of this mechanism during sodium loading is not known.

The effects of L-NMMA on renal hemodynamics were enhanced during HS with the greatest impact on RPF. These data suggest that NO is an important part of the renal vascular adaptation to increased dietary sodium in humans. This finding agrees well with studies in rats (7, 30, 35). Autoregulatory adjustments to changes in systemic perfusion could partly explain the observed differences in this study. However, the autoregulatory response in the renal circulation to systemic NO inhibition has been studied in a rat model with controllable renal perfusion pressure (RPP) (30). This study showed that the enhanced response to L-NMMA during HS intake is further amplified if RPP is kept constant. In addition, the autoregulatory mechanism by itself is probably not influenced by NO (3). Moreover, the main events in the systemic circulation after L-NMMA occurred only within the first 40-50 min of the experiments, whereas the effect on renal hemodynamics was evident within 150 min after injection. This adds to the notion that the renal circulation is particularly sensitive to NO regulation. Thus we find it most likely that our observations on RPF and RVR are due to an increased influence of NO in the renal circulation during HS intake.

NO modulates renal hemodynamics through several mechanisms. NO is synthesized directly in the renal vasculature. Immunohistochemical studies have shown expression of constitutive NOS in glomerular capillaries, afferent and efferent arterioles, medullary vasa recta, and intrarenal arteries (2). With the use of PCR techniques, expression of inducible NOS has also been suggested in renal vessels (23). Sodium loading leads to fluid retention and increased blood volume, thereby increasing shear stress on the endothelial cells, which is a potent stimulus for NO synthesis. There is, however, still no direct evidence of increased vascular NO release or vascular NOS expression during sodium loading in human or animal models. However, in crude renal medullary preparations of rats, it has been shown that the expression of all subtypes of NOS is greatly increased during sodium loading (20). The significance of bradykinin-induced NO synthesis during sodium loading has been investigated recently. HS intake in mice lacking the gene encoding for the bradykinin B2 receptor develop hypertension, accompanied by intense renal vasoconstriction (1). Thus kinin-induced NO synthesis during sodium loading seems to occupy a key role in this adaptory process. Another main mechanism by which NO could regulate renal hemodynamics is through modulation of the tubuloglomerular feedback (TGF) response, which is controlled by the macula densa region. The TGF mechanism elicits a preglomerular vasoconstriction reducing single-nephron GFR in response to increased delivery of sodium chloride at the macula densa cells of the distal tubule. A relative blunting of the TGF response is probably an important part of the renal adaptation to increased sodium intake. As proposed by experiments by Wilcox et al. (37) NO synthesis in the macula densa is stimulated by increased solute transport. In further studies, these investigators have shown that increased dietary sodium attenuates the TGF response by enhancing the synthesis (or effect) of NO in the juxtaglomerular apparatus of rats (36). In this model, the influence of NO on TGF is abolished during LS intake. Considering these studies, our results on renal hemodynamics (and sodium excretion) could be explained partly by a potentiated TGF response of NOS blockade during HS intake, which is abolished during LS intake.

Sodium excretion is profoundly affected by NO. Systemic or intrarenal NO inhibition in animals and humans leads to decreased sodium excretion (4). Even intramedullary administration of NO inhibitor can be shown to elicit antinatriuresis (21). In the present study, we show that the relative influence of NO on sodium excretion is diminished or abolished during LS intake in humans. As we have reported on FENa, the results could not solely be explained by changes in renal hemodynamics and glomerular filtration. To our knowledge, this is the first study to demonstrate the relative influence of NO on sodium excretion during different levels of sodium balance in humans. The results implicate that NO plays a significant role in the renal adaptation to increased sodium excretion in humans. It is obvious to speculate that disturbances in this system could lead to a sodium volume-dependent form of hypertension. Indeed, at least one study has shown a presumably decreased ability to increase NO synthesis and sodium excretion in patients with salt-sensitive essential hypertension after stimulation with L-Arg (16). In addition, chronic NO inhibition in laboratory animals in subpressor doses eventually leads to hypertension after sodium loading (29). The mechanisms by which NO affects sodium excretion are highly complex. As mentioned above, the influence of macula densa-derived NO on the TGF response is probably also operating in humans to regulate sodium excretion. In a recent study, we provided evidence that systemic L-NMMA treatment in sodium-replete humans was accompanied by a decreased fractional lithium excretion (4). This finding indicate that NO acts on proximal sodium reabsorption. Indeed, in vitro studies have provided evidence of a direct or cGMP-mediated inhibitory influence of NO on sodium transporters in the proximal part of the nephron but also distally (6, 14, 22, 27, 32).

Systemic NO inhibition was accompanied by a profound decrease in fluid output and CH2O. Assuming minimal vasopressin action on collecting duct water reabsorption (during water diuresis), it may be suggested that fluid delivery out of the proximal tubule was sharply reduced by L-NMMA estimated by the index (V/GFR) × 100 and hence explain a major part of the reduced sodium and water excretion after L-NMMA. However, a distal effect remains evident estimated by the index (CH2O/GFR) × 100. During HS, the decrease in net water excretion occurred without major changes in urinary osmolality (Table 3 and Fig. 3) indicating that the drop in CH2O was accompanied by a proportionate decrease in solute excretion (probably sodium and chloride). During LS, however, the decrease in CH2O was significantly enhanced at a point where the antinatriuretic effect of L-NMMA was abolished and accompanied by a significant increase in Uosm. Thus, at least during LS balance, NO in some way seems to influence urinary diluting ability. However, baseline Uosm was significantly higher during HS. The reason for this finding is not entirely clear but could be attributed to a long-term renal adaptation to HS intake induced by the need for increased solute excretion.

The effects of NO on water excretion is supported to some extent by animal studies. In a careful dose-titration study in rats, it was shown by Lahera et al. (19) that the first significant effect of systemic NO inhibition was water retention occurring before any effect on renal hemodynamics and sodium excretion was evident. In contrast to these findings, this laboratory earlier reported a decreased CH2O in response to nitroprusside infusion (NO-donating substance) in healthy subjects (25). However, the comparison between these studies is impeded by different study designs. The nitroprusside study was mainly concerned with renal hemodynamics during a significant effect of nitroprusside on systemic hemodynamics leading to a sustained drop in systemic blood pressure and activation of the sympathetic nervous system and the renin-angiotensin-aldosterone axis. We cannot deduce the exact mechanism of the water retaining effect of NO inhibition from these data. Plasma levels of arginine vasopressin (AVP) (data not shown) were too low during both conditions to be of any use in the interpretation of these data. Data from in vitro studies have shown that NO exerts an inhibitory signal on the effects of AVP in terms of the renal sensitivity to the water-retaining effect of this hormone (10). The water-retaining effect of NO inhibition could thus be explained by an increased renal effect of AVP unmasked by the removal of NO. Such a mechanism would also explain why the water-retaining effect of L-NMMA is enhanced during LS intake, because it has been shown recently that the renal sensitivity to AVP is enhanced during LS intake in healthy subjects (33). However, it should be emphasized that the present results were obtained during water diuresis with a marked suppresion of AVP levels. Thus it needs to be confirmed whether this finding is true also during a normal physiological diuresis.

NO has been implicated as a regulator of the secretion of many hormones. Much attention has been focused on the influence of NO on renin secretion. In the present study we found, during LS balance, a sustained decrease in plasma renin after L-NMMA that lasted beyond the normalization of systemic blood pressure. This result extends observations to humans from many in vivo animal studies and studies of isolated perfused kidneys that NO inhibition is accompanied by a decreased renin secretion (11). However, controversy exists considering the fact that many in vitro studies have reported a direct inhibitory effect of NO on renin secretion from juxtaglomerular cell preparations (13). The reason for these discrepancies is not clear, but it is most likely that the observed in vivo effects on renin secretion reflects the sum of several mechanisms (15).

In summary, we have shown for the first time in humans that the systemic and renal hemodynamic response to systemic NO synthesis inhibition is enhanced during HS intake and that HS intake is accompanied by indices of increased NO synthesis. We have shown that the regulatory influence of NO on FENa increases with increasing sodium intake, and we have described an effect of NO on water excretion during LS balance. We have proposed this phenomenon to be linked to an interaction with AVP, which has been described recently. We conclude that, in humans, NO seems to play an important role during the adaptation to variations in sodium intake in terms of the regulation of systemic blood pressure and the renal regulation of body fluid homeostasis.

    ACKNOWLEDGEMENTS

We greatly acknowledge the skillfull technical assistance of our laboratory team, including Lisbeth Mikkelsen, Kirsten Tønder, Rikke Andersen, Dorte Ronde, Jane Knudsen, Elsebeth Fibiger and Gitte Paulsen. We also acknowledge the assistance of clinical dietician Lotte Bak-Henriksen (Aarhus University Hospital) for providing the sodium diets.

    FOOTNOTES

This study was supported by grants from The Research Foundation of Aarhus University and The Danish Medical Research Foundation.

Address reprint requests to J. N. Bech.

Received 2 September 1997; accepted in final form 22 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Renal Physiol 274(5):F914-F923
0363-6127/98 $5.00 Copyright © 1998 the American Physiological Society



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