Macula densa (MD) cells express the Na+:H+ exchanger (NHE) isoform NHE2 at the apical membrane which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine if NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild type (NHE2+/+) and NHE2 knockout (NHE2-/-) mice. The number of renin expressing cells in the afferent arteriole in NHE2-/- mice was increased about 2.5-fold using renin immunohistochemistry. Western blotting confirmed approximately 20% higher renal cortical renin content in NHE2-/- mice compared to wild type. No-salt diet for one week significantly increased renin content in NHE2+/+ mice, but the response was blunted in NHE2-/- mice. Renal tissue renin activity and plasma renin concentration were elevated 1.6 and 2-fold, respectively, in NHE2-/- mice, and they exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2-/- kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion and the signaling pathway likely involves MD cell shrinkage, activation of ERK1/2, COX-2 and mPGES, well established elements of MD-PGE2-renin release pathway.