Regulation of renal adenosine excretion in humans—role of sodium and fluid homeostasis

Nils Heyne1,*, Peter Benöhr2,*, Bernd Mühlbauer2, Ursula Delabar2, Teut Risler1 and Hartmut Osswald2

1 Department of Internal Medicine III, Section of Nephrology and Hypertension, and 2 Department of Pharmacology and Toxicology, University of Tübingen, Germany

Correspondence and offprint requests to: Nils Heyne, MD, Department of Internal Medicine III, Otfried-Müller Str. 10, University of Tübingen, D-72076 Tübingen, Germany. Email: nils.heyne{at}med.uni-tuebingen.de



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Adenosine is a vasoactive metabolite of ATP hydrolysis that is involved in the regulation of renal haemodynamics, tubular reabsorption and renin release. Elevated tissue levels are found under conditions of increased metabolic load, ischaemia or renal injury. Urinary adenosine excretion (EADO) may therefore provide a sensitive marker of renal functional impairment in allograft rejection and kidney disease. To provide a basis for evaluation of EADO in clinical settings, we investigated, in an intra-individual, crossover clinical trial the physiological variability and regulation of EADO in response to altered sodium and fluid balance.

Methods. Twelve healthy volunteers were randomized to normal (ad libitum), low (<5 g/day) or high (supplementation of 100 mg/kg/day) sodium chloride diets for 8 days prior to assessment of renal haemodynamics and tubular function in standard clearance investigations. Following baseline periods, fluid homeostasis was altered independently by acute oral water load. EADO was determined in 24 h urine collections and during clearance investigations.

Results. Mean EADO in humans was 3.2±0.2 µmol/ 24 h during euvolaemia and normal sodium intake. A weak correlation was found between sodium load and EADO. In clearance experiments, variation in EADO was <1.3-fold, despite profound alterations in sodium intake. EADO was independent of urinary flow rate. Renal haemodynamics were not significantly altered by dietary regimen or by acute volume load.

Conclusion. In summary, the physiological variability of EADO is remarkably small in humans. We demonstrate that even profound alterations in sodium and fluid homeostasis do not significantly affect EADO. These data provide a basis for evaluation of elevated EADO as a marker of renal injury in various clinical settings.

Keywords: adenosine; fluid and electrolyte homeostasis; natriuresis; sodium intake; urinary adenosine excretion



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Adenosine is a vasoactive metabolite of ATP hydrolysis, derived from the intracellular degradation of AMP by 5'-nucleotidase or via hydrolysis of S-adenosylhomocysteine [1]. In the rat normoxic kidney, whole tissue adenosine content is ~4–5 nmol/g wet weight [2], and interstitial fluid concentrations of ~200 nmol/l have been reported [3]. Interstitial adenosine is metabolized rapidly by cellular uptake and degradation by adenosine deaminase or via phosphorylation to AMP by adenosine kinase. With a plasma half-life of 1–3 s, adenosine is a tissue hormone acting in a paracrine manner.

In the kidney, adenosine is involved in the regulation of renal haemodynamics, tubular reabsorption of fluid and solutes, and in renin release. In contrast to other vascular beds, adenosine induces vasoconstriction in the kidney, thereby coupling renal perfusion to the metabolic rate of the organ [4,5]. Most of the oxygen consumption in the kidney is required for transepithelial sodium reabsorption. To limit renal energy expenditure, afferent arteriolar tone and glomerular perfusion are regulated by a negative feedback mechanism (tubuloglomerular feedback, TGF), dependent upon the tubular load of fluid and solutes. An increase in early distal flow rate and chloride concentration at the macula densa site results in an activation of TGF, with subsequent afferent arteriolar vasoconstriction [5], and a reduction in glomerular filtration rate (GFR) and tubular load. Substantial experimental evidence indicates that adenosine is involved in signal transmission and is a principal mediator of the TGF response [4,5]. The concept of adenosine acting as a homeostatic metabolite in metabolic control of organ function has been discussed extensively in the literature [6,7].

Experimental investigations demonstrated elevated tissue adenosine concentrations during conditions of increased metabolic load [4], renal ischaemia [2] or drug-induced nephrotoxicity [9]. Urinary adenosine content varies with altered interstitial adenosine concentrations [10]. Therefore, urinary adenosine should reflect energy expenditure as well as renal functional deterioration following ischaemia or direct cellular damage and may provide an easily accessible marker for the diagnosis and quantification of renal injury.

Clinical investigations have shown an increase in urinary adenosine excretion (EADO) following drug-induced nephrotoxicity [11,12] or during acute renal allograft rejection [13]. However, interpretation of these data consistently remained difficult due to the lack of reference values in humans and to the frequent and substantial fluid and electrolyte disorders encountered in these clinical settings.

The present trial therefore investigated the physiological variability and regulation of EADO in response to altered sodium and fluid balance in healthy subjects. Such data provide a basis for the clinical evaluation of EADO in renal allograft function, drug-induced nephropathy and kidney disease.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Study population
Twelve healthy volunteers (six male, six female, aged 21–32 years, median age 25.5 years) were enrolled in the study on an out-patient basis. Subjects were normotensive, non-obese (mean body mass index 23.9±0.9), non-smokers, without medical history of renal, endocrine or cardiovascular disease, and received no medication. Approval by the local ethics committee and written informed consent from each subject was obtained.

Study design and protocol
The study was designed as an intra-individual, randomized crossover trial. Subjects were studied on three occasions, following varied 8 day dietary sodium regimen, separated by 7 day wash-out periods. Volunteers were randomly allocated to different sequences of normal, low or high sodium intake to compensate for time and carryover effects. Sodium intake was either ad libitum, dietetically restricted to <5 g/day or ad libitum plus supplementation of 100 mg/kg/day encapsulated sodium. Compliance with the dietary regimen was monitored by measuring sodium excretion in consecutive 24 h urine collections on the last 2 days of pre-treatment. Following each dietary regimen, renal haemodynamics and tubular function were assessed in clearance investigations. EADO was determined in 24 h urine collections and during clearance investigations.

Clearance investigations
For clearance investigations, inulin and para-aminohippurate (PAH) were used as marker substances for calculation of GFR and renal plasma flow (RPF), respectively. Marker substances were dissolved in isotonic saline, except in subjects on low sodium intake, where isoosmolar (5%) glucose solution was used in order to maintain sodium depletion. Following equilibration, three 30 min baseline clearance periods were performed, during which fluid losses where substituted orally (v/v) by medicinal water. Subsequently, an oral water load of 1500 ml was given within 30 min and another four clearance periods were performed.

Urine samples were collected at the end of each clearance period by spontaneous voiding. Aliquots for determination of urinary adenosine concentrations were acidified with sulfosalicylic acid (10 mg/ml). Blood samples were drawn at mid-period.

Concentrations of inulin and PAH in plasma and urine were determined by colorimetric assay. Clearances were calculated according to standard formulae and normalized for a body surface area of 1.73 m2 for GFR and RPF. Filtration fraction was expressed as the ratio of GFR over RPF; renal blood flow (RBF) was calculated as RPF divided by (1 – haematocrit). Free water clearance was calculated as urinary flow rate minus osmolar clearance. Plasma and urinary electrolyte concentrations were determined by flame photometry. Mean daily sodium intake was calculated from the average sodium excretion of two consecutive 24 h urine collections. Plasma renin activity was determined by radioimmunoassay and urinary adenosine concentrations were measured by HPLC [14].

Data analysis
Data are expressed as means±SEM. Differences among groups were compared by ANOVA, followed by Bonferroni post hoc tests. Correlations were assessed by linear regression analysis. A P-value <0.05 was considered significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Efficacy of dietary sodium regimen
The effects of sodium diets on blood pressure, fluid and electrolyte homeostasis and plasma renin activity are shown in Table 1. Sodium intake was as low as 1.2±0.3 g/day under sodium restriction and attained 15.6±1.0 g/day under sodium supplementation. As expected, plasma renin activity was elevated during sodium restriction and suppressed during high sodium intake.


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Table 1. Effects of differing sodium intake on mean arterial pressure (MAP), serum haematocrit (HCT), plasma renin activity (PRA), 24 h urine volume (UV), and sodium (ENA), potassium (EK) and adenosine excretion (EADO)

 
No differences among dietary regimen were observed regarding mean arterial pressure, serum haematocrit or 24 h urine volume. Adherence to the dietary regimen was strict, producing a >14-fold variation in 24 h urinary sodium excretion. Sodium restriction markedly increased urinary potassium excretion.

Effects of sodium intake on 24 h urinary adenosine excretion
Mean 24 h EADO in healthy euvolaemic volunteers on normal sodium intake was 3.21±0.21 µmol/24 h (Table 1). Adenosine excretion was lower under sodium restriction (2.62±0.20 µmol/24 h) and was elevated under high sodium intake (3.92±0.26 µmol/24 h; P<0.05 vs low sodium intake). Individual data revealed a weak positive correlation (r2 = 0.38, NS) between EADO and urinary sodium excretion.

Baseline clearance investigations
Findings from baseline clearance investigations following 12 h of water deprivation are presented in Table 2. GFR tended to be higher following sodium supplementation; however, no significant differences in renal haemodynamics were observed among the pre-treatments. Baseline urine flow was comparable and independent of sodium intake. Fractional sodium excretion was significantly lower under sodium restriction and elevated during high sodium intake. Clearance of osmolytes changed accordingly, with a negative free water clearance at baseline under sodium supplementation. As with the 24 h urine collections, a small effect of sodium diets on baseline EADO was observed. However, no significant differences in EADO were found when expressed per ml of normalized GFR.


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Table 2. Effects of differing sodium intake on renal haemodynamics, tubular function and urinary adenosine excretion at baseline and following acute oral volume load

 
Effects of acute volume load
Following baseline, an acute oral water load was applied to independently alter fluid homeostasis. Acute volume load did not alter renal haemodynamics from baseline levels (Table 2). Urine flow was significantly increased, comparable in magnitude and time course under conditions of normal and high sodium intake. Volunteers on sodium restriction displayed a slightly delayed and less pronounced response. As expected, diuresis was characterized by a substantial and significant increase in free water clearance, with only small changes in urinary electrolyte excretion. EADO was unchanged and independent of urine flow in all groups (Table 2 and Figure 1).



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Fig. 1. Correlation between urinary adenosine excretion (EADO) and urine flow (UF) during different dietary sodium regimen, prior to (closed symbols) and following acute volume load (open symbols): circles normal, upside down triangles low and triangles high sodium intake.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present trial establishes reference values for EADO in humans and describes the physiological variability and regulation of EADO in response to changes in fluid and electrolyte balance. These data provide a basis for evaluation of elevated EADO as a marker of renal injury in different clinical settings.

The amount of adenosine excreted with the urine is determined by various processes, including (i) glomerular filtration of adenosine; (ii) intratubular adenosine generation from different precursors; (iii) proximal tubular reabsorption; and (iv) the direct release of adenosine from tubular epithelium. From the present data, an estimate of the quantitative contribution of these sources to urinary adenosine can be made.

In the kidney, plasma adenosine is freely filtered. With an arterial plasma adenosine concentration of ~80 nmol/l in humans [15] and a mean GFR of 95 ml/min, as assessed in this trial, filtered adenosine will amount to ~7.6 nmol/min. We observed a mean EADO of ~3.2 nmol/min in euvolaemic volunteers on normal sodium intake (Table 2), indicating a tubular reabsorption of ~50% of filtered adenosine. A similar fractional clearance of exogenous tracer adenosine has been reported previously in dogs [10]. In addition, urinary adenosine may also be derived from intratubular conversion of its nucleotide precursors. Ectoenzymes, required for extracellular adenosine generation, such as ecto-phosphodiesterase and ecto-5'-nucleotidase, are specifically distributed at proximal and distal tubular sites [16]. However, the contribution of intratubular adenosine generation to urinary adenosine content is small under physiological conditions.

In the proximal tubule, adenosine is actively reabsorbed via a sodium gradient-driven nucleoside transporter. Experimental data have shown sodium loading to increase interstitial and urinary adenosine concentrations in the rat [17,18]. In humans, the effect of sodium loading on EADO was small. Despite a >9-fold variation in FENa, a <1.3-fold variation in EADO was observed. Nevertheless, this finding may reflect enhanced adenosine generation under increased metabolic load. In addition, sodium loading reduces the luminal transepithelial sodium gradient, thereby diminishing the efficiency of sodium/adenosine co-transport in proximal tubular cells [19]. Importantly, the increase in EADO was independent of urinary flow rate. Passive back-diffusion of adenosine is therefore unlikely to occur alongside the tubule. Within urinary space, degradation of adenosine is slow and high urine flow rates make it unlikely that urinary degradation of adenosine significantly affected EADO in our study. We therefore provide evidence that under physiological conditions, filtered adenosine represents the major fraction of urinary adenosine in humans, and that ~50% of filtered adenosine is subject to tubular reabsorption. Physiological variations in EADO do not exceed 30% in response to sodium loading and are independent of fluid homeostasis.

Under pathophysiological conditions, however, the situation differs significantly. Depending on cytosolic free adenosine concentrations, a variable amount of adenosine may be released directly into the tubule by cellular leakage. Adenosine metabolism is profoundly altered in acute and chronic inflammation [20], and a several fold increase in cytosolic concentrations of adenosine and its nucleotide precursors has been demonstrated following ischaemia [2] or drug-induced nephrotoxicity [9]. To date, only few clinical trials have adressed urinary adenosine excretion. Katholi and co-workers [11] demonstrated a significant increase in EADO following radiocontrast media administration, which paralleled renal functional impairment. Comparable results were obtained by Baggott et al. [12] in patients receiving methotrexate therapy. Recent data from our department in >100 kidney transplant recipients demonstrated a 2- to 5-fold increase in mean EADO during severe cyclosporin A overdosage or during bioptically verified acute renal allograft rejection [13] as compared with stable allograft function. The observed increments exceeded by far the physiological variability of EADO as shown in the present investigations, and cannot be attributed to concurrent fluid or electrolyte disorders.

These findings support the concept that elevated EADO reflects renal functional impairment due to unfavourable metabolic conditions, inflammation or cellular injury. Clearly, further clinical trials will be required to evaluate the diagnostic and prognostic value of EADO in these settings. As a basis for future clinical trials, the present investigations establish reference values and describe the physiological range of EADO in humans.



   Acknowledgments
 
The authors gratefully acknowledge the skillful assistance of Walter Beer, Janina Smykowski, Antje Raiser, Gerd Luippold and Markus Hanesch. N.H. was supported by the Federal Ministery of Education and Research (BMBF 01 EC 9405).

Conflict of interest statement. None declared.



   Notes
 
*N. Heyne and P. Benöhr contributed equally to this work. Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 17.11.03
Accepted in revised form: 2. 6.04





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