Ontogeny of NHE8 in the rat proximal tubule

Abstract

Proximal tubule bicarbonate reabsorption is primarily mediated via the Na(+)/H(+) exchanger, identified as NHE3 in adults. Previous studies have demonstrated a maturational increase in rat proximal tubule NHE3 expression, with a paucity of NHE3 expression in neonates, despite significant Na(+)-dependent proton secretion. Recently, a novel Na(+)/H(+) antiporter (NHE8) was identified and found to be expressed on the apical membrane of the proximal tubule. To determine whether NHE8 may be the antiporter responsible for proton secretion in neonates, the present study characterized the developmental expression of NHE8 in rat proximal tubules. RNA blots and real-time RT-PCR demonstrated no developmental difference in the mRNA of renal NHE8. Immunoblots, however, demonstrated peak protein abundance of NHE8 in brush border membrane vesicles of 7- and 14-day-old compared with adult rats. In contrast, the level of NHE8 expression in total cortical membrane protein was higher in adults than in neonates. Immunohistochemistry confirmed the presence of NHE8 on the apical membrane of the proximal tubules of neonatal and adult rats. These data demonstrate that NHE8 does undergo maturational changes on the apical membrane of the rat proximal tubule and may account for the Na(+)-dependent proton flux in neonatal proximal tubules.

Fig. 1

Maturational changes in Na⁺/H⁺ exchanger [NHE8 (A) and NHE3 (B)] mRNA abundance. RNA blots with 5 μg of poly(A)⁺ RNA from rat renal cortex are shown for 1-, 7-, 14-, and 28-day-old (1D, 7D, 14D, and 28D) and adult (AD) rats. GAPDH mRNA was used to ensure equal loading of lanes. There was no significant maturational change in NHE8 mRNA expression. There was a significant maturational increase in NHE3 expression. Values are means ± SE of 3 measurements in each age group. *P < 0.05 vs. 26D and AD.

Fig. 2

NHE8 (A) and NHE3 (B) mRNA abundance. cDNA synthesized from rat renal cortex total RNA was analyzed by real-time RT-PCR, with 28S rRNA as a control. Results are consistent with RNA blot analysis. There was no significant maturational change in NHE8 expression. There was a significant maturational increase in NHE3 expression. Values are means ± SE of ≥7 measurements in each age group. *P < 0.05 vs. 26D and AD.

Fig. 3

Maturational changes in NHE8 (A) and NHE3 (B) protein abundance in renal brush border membrane vesicles (BBMV). NHE8 and NHE3 protein abundance relative to β-actin was determined by immunoblots of 50 μg of rat renal BBMV (NHE8 and NHE3 were ∼80 kDa). Developmental pattern of NHE8 is the reciprocal of developmental pattern of NHE3. Equal loading of samples was verified by β-actin. Values are means ± SE of ≥5 measurements in each age group. *P < 0.05 vs. 1D and AD. +P < 0.05 vs. 26D. #P < 0.05 vs. 26D and AD. ˆP < 0.05 vs. 1D.

Fig. 4

NHE8 protein abundance in rat cortical total protein. Immunoblots of cortical total protein demonstrate highest relative abundance of NHE8 in adult. Equal loading of samples was confirmed using β-actin. Values are means ± SE of 4 measurements in each age group. *P < 0.05 vs. AD.

Fig. 5

NHE8 abundance in rat cortical total membrane protein relative to β-actin. Immunoblots of rat cortical total membrane protein (including apical, basolateral, and intracellular membranes) show highest relative abundance of NHE8 in adult. Values are means ± SE of 4 measurements in each age group. *P < 0.05 vs. AD.

Fig. 6

Immunohistochemistry of NHE8 in neonatal and adult rat proximal tubule. Neonatal (10 days) and adult rat kidneys were fixed by perfusion, and sections were probed with anti-NHE8 with fluorescein secondary antibody for NHE8 protein (green), rhodamine-phalloidin for actin (red), and 4,6-diamidino-2-phenylindole (DAPI) for nuclei (blue). Images are representative of 4 independent sets of animals that showed similar results.

Fig. 7

Immunohistochemistry of NHE8 in 1-day-old (A), 10-day-old (B), and adult (C) rat kidney. NHE8 is costained with villin (brush border), Lotus tetragonolobus agglutinin (LTA, proximal tubule), and Na⁺/Ca²⁺ exchanger (NCX-1, distal tubule). Superficial cortex is shown at top of the low-magnification figures stained for NHE8 alone. Nephrogenic zone is still present in 1-day-old neonates (A, left). In low magnification of developing kidney, NHE8 staining is most intense, but not limited to proximal tubule. Although it is likely that this staining represents background, we cannot rule out expression in other nephron segments. NHE8 staining appears to be heterogeneous on apical membrane of proximal tubules, and NHE8 labeling may be limited to certain segments of proximal tubule in neonatal and adult kidney. Scale bars, 50 μm.