Localization and functional characterization of Na+/H+ exchanger isoform NHE4 in rat thick ascending limbs

The Na+/H+ exchanger NHE4 was cloned from a rat stomach cDNA library and sh own to be expressed predominantly in the stomach and less dramatically in t he kidney. The role and precise localization of NHE4 in the kidney are stil l unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification o f thick ascending limbs and collecting ducts, respectively. The results ind icate that NHE4 is highly expressed in basolateral membranes of thick ascen ding limb and distal convoluted tubule, whereas collecting ducts from corte x to inner medulla and proximal tubules showed weaker basolateral NHE4 expr ession. Western blot analysis of NHE4 in membrane fractions prepared from t he inner stripe of the outer medulla revealed the presence of a 95-kDa prot ein that was enriched in basolateral membrane vesicles isolated from medull ary thick ascending limbs. The inhibition curve of H+-activated Na-22 uptak e by 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was consistent with the presen ce, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 muM. Kinetic analyses showed that the extracellular Na dependence of NHE4 activity followed a simple hyp erbolic relationship, with an apparent affinity constant of 12 mM. Intraves icular H+ activated NHE4 by a positive cooperative mechanism. NHE4 had an u nusual low affinity for intravesicular H+ with a half-maximal activation va lue of pK 6.21. We conclude that NHE4, like NHE1, is expressed on the basol ateral membrane of multiple nephron segments. Nevertheless, these two prote ins exhibited dramatically different affinities for intracellular H+, sugge sting that they may play distinct physiological roles in the kidney.