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
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Abstract |
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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
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Introduction |
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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.
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Subjects and methods |
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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.
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Results |
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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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Discussion |
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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.
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Acknowledgments |
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Conflict of interest statement. None declared.
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Notes |
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References |
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