Effects of extracellular nucleotides on renal tubular solute transport

Abstract

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y(1) receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K(+) channels in mouse collecting duct, apparently through stimulation of P2Y(2) receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y(2) receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.

Fig. 1

Current information on P2 receptor subtypes along the rat nephron (native tissue only). P2 receptor subtypes were identified using immunohistochemistry and/or western blotting. Where possible, apical (a), basolateral (b) or intracellular (intra) location is indicated. Information is taken from references [4, 5, 9, 10, 36, 37]

Fig. 2

Stimulation of apical P2Y₁ receptors inhibits bicarbonate reabsorption in rat proximal convoluted tubule. a Expression of P2Y₁ receptor protein in brush-border membrane vesicles harvested from rat proximal tubule. Total protein (30 μg/lane) from three animals (C1, C2, C3) was probed using an antibody raised against residues 242–258 of the human P2Y₁ receptor protein (Alomone Labs, Israel); preabsorption of the antibody with this antigen prevented detection (right-hand side of the trace). Western blot is courtesy of Dr. Joanne Marks. b Effect of adenine nucleotides on bicarbonate reabsorption in the microperfused rat proximal tubule in vivo, expressed as percentage of the control flux measured in the same tubule. The inhibitory profile of naturally occurring adenine nucleotides is consistent with a P2Y₁ receptor-mediated response. This was confirmed using the selective agonist 2MeSADP and specific antagonist MRS2179. c The effect of 2MeSADP was not additive to that of EIPA, suggesting an NHE-3-mediated response. Inhibition of bicarbonate flux by P2Y₁ receptor activation was blocked by either U73122 or H89, indicating involvement of phospholipase C and protein kinase A, respectively. Data are taken from Bailey [14]. *P < 0.05, **P < 0.01 against paired control values

Fig. 3

Effect of ATP on low-conductance K⁺ channels in the apical membrane of mouse cortical collecting duct. Patch-clamp techniques were applied to split-open tubules. a Channel recording showing that addition of ATP (100 µM) to the bathing solution (grey band) caused closure of the K⁺ channel, an effect that was reversible. b Dose–response curve for the effect of ATP on K⁺ channel open probability. The action of ATP was blocked by the P2 receptor antagonist suramin and was mimicked by UTP, whereas αβMeATP and 2MeSATP were without effect on K⁺ channel activity, suggesting the involvement of P2Y₂ receptors. Taken, with permission, from Lu et al [40]

Fig. 4

Inhibition of collecting duct sodium reabsorption by luminally applied nucleotides. a Effect of luminal application of ATP on short-circuit current (SCC) and [Ca²⁺]_i (assessed as the fura-2 fluorescence emission ratio at 345/380 nm excitation) in mouse cortical collecting duct perfused in vitro. The mice had been kept on a low-sodium diet (to enhance ENaC activity). The SCC was shown to be amiloride-sensitive and was therefore taken to represent ENaC-mediated Na⁺ transport. The effect of ATP was mimicked by UTP. Taken, with permission, from Lehrmann et al. [46]. b Effect of intraluminal nucleotides on collecting duct ²²Na absorption in sodium-restricted rats, in vivo. Late distal tubules were perfused with artificial tubular fluid containing ¹⁴C-inulin and ²²Na, and urinary recoveries were monitored. Each distal tubule was perfused twice, first with a control perfusate, then with a P2 agonist. The broad-spectrum agonist ATPγS significantly increased urinary ²²Na recovery, but the P2Y₁ agonist 2MeSADP and the P2Y₂/P2Y₄ agonists Cp₄U and Ap₄A were without effect. Data are taken from Shirley et al. [47]