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1 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA
2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
3 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: peter.aronson{at}yale.edu.
In situ hybridization studies demonstrated that NHE8 is expressed in kidney proximal tubules. Although membrane fractionation studies suggested apical brush border localization, precise membrane localization could not be definitively established. The goal of the present study was to develop isoform-specific NHE8 antibodies as a tool
to directly establish the localization of NHE8 protein in the kidney by immunocytochemistry. Towards this goal, two sets of antibodies that label different NHE8 epitopes were developed. Monoclonal antibody 7A11 and polyclonal antibody
Rab65 both specifically labeled NHE8 by Western blotting as well as by immunofluorescence microscopy. The immunolocalization pattern in the kidney seen
with both antibodies was the same, thereby validating NHE8-specificity. In particular, NHE8 expression was observed on the apical brush border membrane of all proximal
tubules from S1 to S3. The most intense staining was evident in proximal tubules in the deeper cortex and medulla with a significant but somewhat weaker staining in superficial proximal tubules. Co-localization studies with 
-glutamyltranspeptidase and megalin
indicated expression of NHE8 both on the microvillar surface membrane and the coated pit region of proximal tubule cells, suggesting that NHE8 may be subject to endocytic retrieval and recycling. Although co-localizing in proximal tubule with NHE3, no significant alteration in NHE8 protein expression was evident in NHE3-null mice. We
conclude that NHE8 is expressed on the apical brush border membrane of proximal tubule cells where it may play a role in mediating or regulating ion transport in this
nephron segment.
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