Immunolocalization of NHE8 in rat kidney

Abstract

In situ hybridization studies demonstrated that Na+/H+ exchanger 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. Toward 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. Colocalization studies with gamma-glutamyltranspeptidase and megalin indicated expression of NHE8 on both 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 colocalizing in the 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.

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…In renal proximal tubules, brush border NHE8 protein expression decreases with maturation although the total amount of NHE8 in renal cortical membranes is higher in the adult compared to the neonate. Furthermore, immunostaining of adult proximal tubules revealed NHE8 in coated pit regions in addition to brush borders [53]. NHE8…”

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation

“…NHE8 localizes predominantly to mid-to transGolgi stacks, where it may alkalinize the luminal pH (17). NHE8 was also reported to be present on multivesicular bodies (23) and somewhat surprisingly at the apical brush-border membrane of epithelial cells of the intestine and renal proximal tubules (22,24,25). Nhe8 Ϫ/Ϫ mice show impaired colonic mucin synthesis and bicarbonate secretion (26,27), a dysfunction of the retinal pigment epithelium (28,29), and male infertility (28,30).…”

Na+/H+ Exchangers in Renal Regulation of Acid-Base Balance

“…35 NHE8 function may be similar to NHE3 (Fig 4), but its precise role in the maintenance of acid-base balance remains to be explored. NHE8 protein expression is not increased in NHE3 knockout mice, 33 suggesting that it may not be regulated by acid-base status the way NHE3 is. 36 Transcripts of the more ubiquitous intracellular NHE isoforms are present in the kidney but their antigenic localization and functional characterization have not been performed.…”

“…NHE4 knockout mice have no documented overt disease phenotype to date. 31 NHE8, the most recently identified renal NHE isoform, is expressed at the luminal membrane of the proximal tubule 32,33 (Fig 3). In the neonate, proximal tubule NHE activity is relatively high despite very low NHE3 antigen levels.…”

“…34 NHE8 expression is higher in young animals and lower in adults, suggesting a potential role for NHE8 during early development. 33 The clear presence of apical NHE activity in the double NHE2 and NHE3 knock-out mice may be a reflection of NHE8. 35 NHE8 function may be similar to NHE3 (Fig 4), but its precise role in the maintenance of acid-base balance remains to be explored.…”