Na⁺-H⁺ exchangers (NHEs) are ubiquitous proteins with a very wide array of physiological functions. The NHE3 isoform plays a crucial role in Na⁺ reabsorption, acid-base homeostasis and volume regulation and has been implicated in hypertension. Two basic regulatory mechanisms are involved in NHE3 regulation: (a) changes in turnover number and (b) changes in trafficking. The aim of this study was to evaluate whether changes in renal sympathetic nerve activity (RSNA) could modulate retraction of NHE3 from the microvillar tips to the intermicrovillar clefts and thence to endosomes. The effect of RSNA on the activity of the exchanger was also assesed. In vivo studies were performed on chlorolose/urethane (1ml, 16.5:250mg/ml, I.P.) anaesthetised rats. Cannulae were placed in the femoral artery to measure blood pressure and in the femoral vein to infuse saline and inulin. Using flank incisions, the right ureter was cannulated; the left kidney was exposed, its ureter cannulated and it was subjected to surgical denervation. After 2 h stabilisation, two 15 min clearances were undertaken, the kidneys were excised and placed on ice. The cortices were dissected free and brush border membranes were obtained using Mg²⁺ precipitation and differential centrifugation (Weinman et al. 1987). The membranes were subjected to SDS-PAGE and, following Western blotting, NHE3 was quantified and identified. Exchange activity was assayed using the acridine orange (AO) fluorescent method in apical membrane vesicles also prepared by Mg²⁺. Exchange activity was measured as the reappearance of AO from acid-loaded vesicles (Besse-Eschmann et al. 2004). Specific activity was expressed as the slope of the initial Na⁺-dependent fluorescent increase. Data (means±S.E.M.) were subjected to Student’s t test and significance was taken when P<0.05. Western blot analysis showed that NHE3 abundance was reduced 3-fold after denervation in relation to the innervated kidney (n=8). There was significantly higher (P<0.01) urinary Na⁺ excretion in the denervated kidney (3.17±0.70mmol/min/kg) as opposed to the innervated kidney (1.16±0.24mmol/min/kg). Fractional Na⁺ excretion was also significantly elevated (P<0.01) in the denervated kidney (0.67±0.15 vs.0.26±0.04%). GFR was similar in both the innervated and denervated kidneys at 2.99±0.24 and 3.34±0.24ml/mg/kg, respectively. There was a 17% decrease in NHE3 activity in the denervated kidney. This could possibly be explained by a RSNA-mediated retractionof exchangers to intermicrovillar cleft and endosomal regions. These data demonstrate that renal denervation decreases apical membrane NHE3 abundance and activity, independently of renal haemodynamics.