Evidence is accumulating that extracellular nucleotides may play a role in the regulation of renal water and electrolyte excretion. Nucleotide (P2) receptors are present in both basolateral and apical membranes of renal tubules (Schwiebert & Kishore, 2001), and luminal application of exogenous ATP or its analogues has been found to modulate transport processes in proximal and distal nephron (M.A. Bailey, personal communication; Lehrmann et al. 2002). However, to date, no values for luminal concentrations of endogenous ATP in vivo have been reported, so the extent to which apical P2 receptors are activated under physiological conditions is unknown. In the present study we have therefore used micropuncture to collect fluid from rat proximal tubules in an attempt to quantify endogenous ATP.

Adult male Sprague-Dawley rats (n = 7) were anaesthetised with sodium thiopentone (LINK Pharmaceuticals; 100 mg kg^-1, I.P.), prepared surgically for micropuncture of the left kidney, and infused I.V. with 0.9% NaCl solution (4 ml h^-1). Sharpened glass micropipettes were used to collect fluid from mid-proximal convoluted tubules of superficial nephrons. Each collection lasted 4 min, and samples of the tubular fluid (80 nl) were then deposited in ice-cold water (50 µl) to halt ATP degradation; 10 such samples were pooled in the same diluent, then frozen prior to analysis. ATP concentration was measured using the luciferin-luciferase enzyme reaction (BioThema AB). An assessment was also made of soluble nucleotidase activity in proximal tubular fluid: ATP standard solutions (12 nl) were mixed (under oil) with 100 nl of tubular fluid; the reaction was stopped 5-40 min later. At the end of each experiment, the rat was killed with an overdose of anaesthetic.

In five pooled samples, the ATP concentration was 267 ± 24 nmol l^-1 (mean ± S.E.M.). The half-life of ATP in mid-proximal tubular fluid was found to be 36 min (95 % confidence intervals: 31-43 min) at room temperature.

Taking account of this evidence for soluble nucleotidases in tubular fluid, as well as the presence of ectonucleotidases in proximal tubular brush-border membranes (Vekaria et al. 2003), it is clear that the measured concentration of ATP represents a minimum value for endogenous ATP in the vicinity of the proximal tubular apical membrane. We suggest that this is consistent with a physiological role for luminal ATP in regulating proximal tubular function.

This work was supported by the National Kidney Research Fund and St Peter’s Trust for Kidney, Bladder and Prostate Research.