INTRODUCTION

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IT HAS BEEN KNOWN for a long time that intensive exercise influences renal function, including changes in hemodynamics, concentration mechanisms, and electrolyte excretion (see, e.g., Ref. 22). During and immediately after exercise, urine flow decreases, which is due to 1) lower renal blood flow and subsequently lower glomerular filtration rate (GFR) and 2) increased reabsorption mechanisms at the distal tubule and medullary collecting duct. These phenomena, i.e., lower GFR and lower water excretion, can be partly explained by increased sympathetic activity. The lower sodium excretion rate during exercise, however, which is indicated by a lower percentage of the filtered sodium excreted by the kidneys and the frequently found lower urine electrolyte concentration and osmolality (2), cannot be satisfactorily explained by any hitherto known hormonal mechanism.

The plasma aldosterone concentration increases during and after exercise as a result of a sympathetically and hemodynamically stimulated increase in renin activity. However, the aldosterone effector kinetics are too slow to have any considerable early effects on tubular electrolyte retention (3, 28).

After 1981, when the atrial natriuretic peptide [ANP-(99126)] was discovered by de Bold and co-workers (4), plasma ANP was also assumed to be an important factor in the exercise-associated regulation of renal sodium retention. In the meantime, many studies have been performed showing that ANP release increases in response to physical exercise in a dose- and time-dependent manner. That is, during longer lasting exercise such as marathon running an initial increase is followed by a return toward basal levels (13). The lower renal sodium excretion during exercise, however, is in contrast to the increased plasma ANP concentrations, excluding ANP as a main factor involved in the regulation of renal function during exercise. Furthermore, during the last few years, physiological ANP concentrations have been shown to have hemodynamic effects but no influence on the regulation of tubular sodium excretion (15, 19).

In 1988, urodilatin [Uro; ANP-(95126)], a member of the family of the natriuretic peptides, was isolated from human urine (27). To date, the mode of its synthesis, processing, and secretion is poorly understood. Because Uro occurs in urine, but could not be detected in human plasma, it is assumed to be secreted by the kidney. Immunohistological studies give further evidence that Uro is localized in the distal tubules in the form of its precursor (12). Uro and ANP share similar functional properties, such as, e.g., diuresis, natriuresis, and vasorelaxation (25). Under physiological conditions, Uro is less inactivated by the kidney endopeptidase EC 24.11 than is ANP (14), explaining the different effects of both natriuretic peptides on the inner medullary collecting duct. Secretion of Uro is mainly regulated by circulatory central filling pressure and by extracellular sodium concentration (see Ref. 15). Thus under many physiological conditions Uro and ANP are stimulated in a similar manner.

To date, no data exist about the behavior of Uro excretion and possible natriuretic effects during physical exercise. Increasing plasma sodium concentration (e.g., Ref. 26) and central venous pressure (17) suggest a stimulation of renal Uro secretion, leading to increased sodium excretion. However, other effects may compensate for these stimuli. It was, therefore, the purpose of this study to investigate the natriuretic peptides Uro and ANP-(99126) and their possible influences on renal sodium excretion during and after exercise. Because there were no published data available about the influence of exercise on the secretion of Uro, we used a 60-min-long bout of exercise at 60% of maximum performance, which is well known to change renal functions.

	METHODS

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Test subjects and exercise protocol. Ten nonsmoking male subjects participated in the study after giving their informed consent. Maximum work capacity and maximum oxygen uptake (Oxycon Sigma, Pulmocard, Iserlohn, Germany) were obtained by an ergocycle test (Excalibur, Lode, Germany) that began at 100 W and increased the workload by 16.6 W every minute until exhaustion. The anthropometric data of the test subjects and the results of the vita maxima tests are presented in Table 1. One week after the initial test all subjects performed a 60-min ergocycle test at 60% of the initially determined maximum performance. All tests were started between 11:00 and 12:00 AM, which was 3 h after the last meal. All subjects were instructed to abstain from physical exercise and to keep to their normal nutritional behavior 1 day before the beginning of the test and during the 24-h observation period. Fluid and nutrient ingestion was restricted 3 h before, and up to 1 h after, the test. Thereafter, the subjects were allowed to drink as much fluid as they had lost during the test period, i.e., 1.4 ± 0.1 liter. Room temperature was kept constant at 21 ± 0.5°C, and relative humidity and ambient pressure were 66 ± 3% and 751 ± 3 mmHg, respectively.

Hormonal measurements. Urinary Uro concentrations were determined by radioimmunoassay as described previously (see Ref. 7). The Uro antibody showed no cross-reactivity with human or rat ANP-(99126), brain natriuretic peptide, C-type natriuretic peptide, or several shorter ANP analogs. The antibody was used in a final dilution of 1:7,500. Synthetic Uro served as a standard (2.8-1,428 pmol/l) and was synthesized by solid-phase peptide-synthesis methods. Purity and peptide content of synthetic Uro were confirmed by capillary zone electrophoresis, amino acid sequencing, amino acid analysis, and mass spectroscopy. ¹²⁵I-labeled Uro was used as tracer (Immunodiagnostic, Bensheim, Germany) with a specific activity of 500 µCi/µg.

Analytic methods. During the exercise period, the arterial blood pressure was measured every 15 min, and the heart rate (Polar-Sport-Tester, Polar, Kempele, Finland) was continuously recorded. In heparinized blood, the hemoglobin concentration was determined by a spectroscopic method (OSM3, Radiometer, Copenhagen, Denmark), hematocrit by microhematocrit centrifugation (20.900 g), and lactic acid concentration by the lactate dehydrogenase method (test kit no. 124842, Boehringer Mannheim). In plasma and urine, the electrolyte concentration ([Na⁺], [K⁺], [Cl]) was measured by ion-sensitive electrodes (EML, Radiometer), osmolality by the freezing-point depression method (Osmometer Röbling, Berlin, Germany), and the creatinine concentration by the Jaffé method (test kit no. 124192, Boehringer Mannheim).

Calculations. Changes in plasma volume from the preexercise values were determined by the equation of Dill and Costill (6)

(1)

where PV is plasma volume, [Hb]_b and [Hb]_a are hemoglobin concentration, and Hct_b and Hct_a are hematocrit values before and after the beginning of exercise, respectively.

Statistics. All results are presented as means ± SE. For statistical analysis of significant changes across time, we used a one-way analysis of variance with repeated measurements. To establish significant differences between two different time points, e.g., variations from the initial value during the observation time, the Bonferroni t-test was used as a follow-up test.

	RESULTS

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Exercise performance data (Table 2). During the 60min-long test period, the workload was adjusted to 60% of the individual maximum performance, corresponding to 207 ± 10 W. This exercise intensity could be maintained during the whole exercise period from all subjects. Heart rate continuously increased during exercise, whereas the systolic blood pressure and plasma lactate concentration remained on a constantly higher level than initial values during the second 30 min of the test.

Plasma volume and electrolytes (Table 3). Water and electrolyte status was markedly influenced during and after exercise. Plasma volume decreased by 8.9% at the end of exercise and was slightly overcompensated 4 and 24 h after the test. Plasma osmolality significantly increased by 8.4 mosmol/kgH₂O, and [Na⁺], [K⁺], and [Cl] increased by 4.0, 1.1, and 3.0 mmol/l, respectively. After 1 h of recovery, all of these quantities had reached the preexercise values.

Renal function (Table 4). Urine volume (ml/min) was significantly decreased up to 4 h after exercise and tended to show lower values up to 24 h after cessation. The creatinine clearance, which was used as an indicator of the GFR, was lowered by >50%, reaching the preexercise values during the first hour of recovery. The fractional excretion rate of sodium and chloride decreased markedly up to 1 h after exercise, whereas the free water clearance showed the opposite behavior, increasing during and up to 1 h after the exercise period.

Plasma ANP concentration and urinary Uro excretion. Plasma ANP concentration and urinary Uro excretion were oppositely affected by physical exercise. Whereas ANP concentration increased from 46.5 ± 5.1 (83.1 ± 13, minute 15; 99.8 ± 3.1, minute 30; n = 5) to 124.1 ± 10.6 pg/ml (Fig. 1), Uro excretion decreased by ~60% during the exercise period from 120.8 ± 16.0 to 49.5 ± 9.8 fmol/min, remained on a lower level (52.5 ± 12.1 fmol/min) for 1 h, and returned to the initial level after 4 h of recovery (Fig. 1; analysis of variance; P < 0.001 for changes in both hormones).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Plasma atrial natriuretic peptide (ANP) concentration and renal urodilatin (Uro) excretion before (rest) and after 60 min of exercise at 60% of maximum performance (end). h, Hour. Significantly different from initial values, * P < 0.05, ** P < 0.01.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Relationship between urinary Uro excretion and fractional sodium excretion rate. Partial correlation analysis: r = 0.603, n = 10 subjects, P < 0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Relationship between plasma ANP concentration and fractional sodium excretion rate. Partial correlation analysis: r = 0.335.

	DISCUSSION

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This study shows that urinary Uro excretion is markedly reduced by exercise effects, i.e., it is oppositely affected compared with plasma ANP concentration. Increasing ANP levels during exercise, as observed here, are well established by a great number of recent studies (e.g., Refs. 13 and 26) and are mostly due to changes in cardiac filling pressure and wall tensions. Uro excretion has not yet been studied under exercise conditions. We therefore use the results of studies with other purposes to discuss our results.

Regulation of Uro excretion. At least three mechanisms are known to lead to changes in urinary Uro excretion.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Relationship between urinary Uro excretion and plasma ANP concentration. Partial correlation analysis: r = 0.505.

Uro and ANP effects. After its discovery by de Bold et al. (4), ANP was assumed to be a potent natriuretic peptide, exerting its effects especially at the inner medullary collecting ducts by formation of the second messenger guanosine 3,5-cyclic monophosphate (cGMP). Meanwhile, ANP could be shown to have considerable hemodynamic effects but not to induce natriuresis in physiological situations. As is demonstrated in Fig. 3, there is a tendency toward a negative correlation between ANP and fractional sodium excretion rate. On the other hand, Uro is positively related to sodium excretion (Fig. 2). This is in accordance with the data of recently published studies describing positive relationships between Uro and fractional sodium excretion rate and a lack of correlation between ANP and fractional sodium excretion rate after dietary salt loading and isotonic volume loading (8, 18). Under in vitro conditions, ANP and Uro exert similar natriuretic effects mediated by the natriuretic peptide A receptors at the medullary collecting duct. In vivo, however, most of the filtered ANP molecules are degraded by the endopeptidase EC 24.11, whereas Uro is more resistant against this enzyme (14). Furthermore, Uro is probably produced at the distal parts of the nephron and is scarcely exposed to this endopeptidase (11), thus acting as a paracrine modulator of tubular electrolyte transport.