The Na+/H+ exchanger isoform 3 (NHE3) facilitates Na+ absorption and H+ secretion. NHE3 is expressed in the intestine, proximal tubule and thick ascending limb of the kidney. Conventional NHE3 knockout mice show severe signs of dehydration or die when deprived of NaCl for 3 days. However, the importance of kidney-specific NHE3 inactivation in this context is unknown. To test the role of kidney-specific NHE3 knockout in Na+ homeostasis we studied tubulus-specific NHE3 knockout mice which have been previously generated by us. NHE3loxlox (Con) and NHE3loxloxPax8Cre (NHE3loxloxCre) mice were randomized and studied at baseline and for 10 days on low (<0.01%) or high (4%) NaCl intake (N=5-7/group). At baseline and the end of each diet regimen blood was collected and analyzed for Na+, K+, aldosterone as well as glomerular filtration rate (GFR) determined via FITC-Sinistrin clearance. Body weight was recorded daily. Under baseline conditions there was no difference between genotypes in body weight, fluid or food intake, plasma Na+ or K+, or blood pH. Plasma aldosterone (259±30 vs 237±28 pg/ml, NS) was not significantly different in NHE3loxloxCre compared to Con mice while urinary pH was significantly elevated (8.1±0.1 vs 7.2±0.1, P<0.05). NaCl intake had no impact on plasma Na+ in Con mice; however, plasma Na+ in NHE3loxloxCre mice was susceptible to the effects of low (-3.9±1.0 mM, P<0.05) and high NaCl (+2.2±0.6 mM, P<0.05) compared to baseline. Low NaCl decreased plasma K+ in Con (-0.5±0.2 mM, P<0.05) but to a significant greater amount in NHE3loxloxCre mice (-1.2±0.1 mM, P<0.05). Aldosterone was not significantly different between Con and NHE3loxloxCre under low (345±96 vs 498±78 pg/ml) and high NaCl intake (60±19 vs 86±20 pg/ml). Possibly as a consequence of impaired proximal tubular Na+ reabsorption and consequently activation of tubuloglomerular feedback, GFR was lower in NHE3loxloxCre mice under baseline conditions (457±20 vs 358±17 µl/min, P<0.05). Low NaCl decreased GFR in Con (-110±13 µl/min, P<0.05) and NHE3loxloxCre (-99±8 µl/min, P<0.05) mice, whereas high NaCl was without effect on GFR in either genotype. No significant differences in blood pressure were detected between Con mice after 10 days on low or high Nacl intake. In contrast, NHE3loxloxCre mice show lower systolic blood pressure under low and high NaCl intake (20 and 10 mmHg, respectively, P<0.05). Of note, blood pressure in NHE3loxloxCre mice was salt-sensitive, with a 10 mmHg increase upon high NaCl diet, while heart rate was not affected by dietary NaCl intake or genotype. The results indicate that maintenance of plasma Na+ requires functional NHE3 in the proximal tubule; otherwise dietary NaCl directly impacts total body NaCl possibly by disturbing glomerulo-tubular balance. In addition, the role of renal NHE3 for overall Na+ homeostasis is less detrimental than previously reported for NHE3 knockout mice.