Extracellular nucleotides act via P2 receptors to modify epithelial Na channel (ENaC)-mediated Na reabsorption in the renal distal tubule (1). Pharmacological profiling and the use of genetically engineered mice suggest an inhibitory role for P2Y2 receptors on ENaC activity (2,3). An in vitro investigation using rat split-open CDs has provided evidence that apical P2X4 receptors may also modulate ENaC activity, and, at a Na concentration that mimics distal tubular fluid, activation of P2X4 by extracellular nucleotides can potentiate ENaC activity (4). There is also evidence that P2X4 may be involved in BP regulation as P2X4 null mice (P2X4-/-) are hypertensive (5). In the present study we investigated the possible role of the P2X4 receptor in the regulation of ENaC activity in P2X4-/- and wildtype littermate controls (P2X4+/+). P2X4+/+ and P2X4-/- mice were maintained on either a standard Na diet (SSD; 0.3%) or a low Na diet (LSD; 0.03%) for 2 weeks. They were then anaesthetised (thiobutabarbital sodium (Inactin), 100mg/kg IP) and surgically prepared for renal clearance studies. After control collections of urine and plasma, mice were given benzamil (1 mg/kg bolus,IV) and further collections were made. Mean arterial (MA) BP was recorded during the experiments. Data are presented as mean±SEM; statistical comparisons were made using ANOVA and post-hoc Bonferonni. MABP was significantly elevated in P2X4-/- mice compared with P2X4+/+ mice on a SSD (95.04±2.3 vs 85.2±2.6mmHg; P=<0.01; n=8 for both). On a LSD MABP was similar in the 2 genotypes: the reduction in dietary Na had no effect on MABP in P2X4+/+ mice but significantly reduced MABP in P2X4-/- mice (87.4±3.2 vs 86.7±2.6mmHg; n=6 for both). On a SSD benzamil administration led to a significant increase in fractional Na excretion (ΔFENa) in both genotypes: values were similar for P2X4+/+ and P2X4-/- mice (1.73±0.25 vs 1.91±0.17%; n=8 for both). On a LSD P2X4+/+ mice showed an increased excretory response to benzamil (expressed as ΔFENa) compared with their response while on a SSD, indicating an increase in ENaC-mediated Na reabsorption (3.72±0.69 vs 1.73±0.25%; P=<0.01; n=6 and 8, respectively); however, P2X4-/- mice did not significantly increase their ΔFENa on a LSD compared to a SSD (1.91±0.17 vs 1.74±0.44%; n=6 and 8 respectively), and had a significantly lower ΔFENa than P2X4+/+ mice on the LSD (1.74±0.44 vs 3.72±0.69%; P<0.01; n=6 for both). The difference in MABP response to the LSD between the P2X4-/- and P2X4+/+ mice suggests that the normally raised BP in P2X4-/- mice is Na-dependent. Moreover, the inability of the P2X4-/- mice to increase their ENaC-mediated Na reabsorption after LSD feeding, as indicated by their response to benzamil, is compatible with the earlier in vitro finding in CDs that at a distal tubule luminal fluid Na concentration P2X4 activation by extracellular nucleotides potentiates ENaC activity.