Increased Na/H antiporter activity has been demonstrated after in vivo chronic metabolic acidosis as well as in vitro acid preincubation of cultured rabbit renal tubule cells. To study the underlying molecular mechanisms of this adaptive increase in Na/H antiporter activity, the present studies examined the effect of low pH media on Na/H antiporter activity and mRNA abundance in cultured renal tubule cells. Na/H antiporter activity was increased by 60% in a mouse renal cortical tubule cell line (MCI'), and by 90% in an oppossum kidney cell line (OKP) after 24 h of preincubation in acid (low [HCO3J) media. The ethylisopropylamiloride sensitivity of the Na/H antiporters were different in these two cell lines (MCI' IC5 = 65 nM; OKP IC5o = 4.5 MM). In MCT cells, Na/H antiporter mRNA abundance measured by RNA blots increased by two-to fivefold after 24 h in low IHCO31 media. Na/H antiporter mRNA abundance was also increased in MCT cells with high CO2 preincubation as well as in rat renal cortex with in vivo chronic acid feeding. In contrast to renal epithelia, acid preincubation of NIH 3T3 fibroblasts led to suppression of Na/H antiporter activity. RNA blots of 3T3 fibroblasts revealed the same size Na/H antiporter transcript as in MCT cells. However, Na/H antiporter mRNA levels were suppressed by acid preincubation. These studies demonstrate differential regulation ofNa/H antiporter activity and mRNA abundance in renal epithelial cells and fibroblasts in response to an acidotic environment. (J.

With the use of degenerate primers based on conserved amino acid sequences in human, rat, and rabbit Na/H exchanger-3 (NHE-3), a polymerase chain reaction product was obtained from reverse-transcribed OKP (a clonal opossum kidney cell line) mRNA and used to screen an OKP cDNA library. The clone obtained predicted an amino acid sequence that was 86% identical to rat NHE-3, 33% to NHE-1, 35% to NHE-2, and 30% to NHE-4. Expression of the corresponding cRNA in Xenopus oocytes induced 22Na uptake with ethylisopropylamiloride. (EIPA) resistance similar to that of the OKP Na/H antiporter. On RNA blot, the cDNA labeled a 9.5-kb transcript whose abundance was increased 2.2-fold by 24-h incubation of OKP cells at pH 7.0 and 2.5-fold by 24-h incubation at pH 6.8. The acid-induced increase in NHE-3 mRNA was detectable at 12 h and increased further at 24 h. Incubation in acid media caused an increase in EIPA-resistant Na/H antiporter activity that preceded the increase in NHE-3 mRNA. In summary, OKP cells express an NHE-3 transcript that encodes an EIPA-resistant Na/H antiporter and is chronically regulated by acid.

To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP

Previous studies have demonstrated that systemic administration of glucocorticoids stimulates proximal tubule acidification in part by increasing Na+/H+ antiporter activity; however, these studies could not exclude the possibility that changes in Na+/H+ antiporter activity were secondary to glucocorticoid-induced hemodynamic changes. The present study examined the effect of dexamethasone on Na+/H+ antiporter activity in quiescent OKP cells. Na+/H+ antiporter activity was assayed as the initial rate of Na(+)-dependent pH recovery from an acid load. Intracellular pH was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Dexamethasone produced a dose- and time-dependent stimulation of Na+/H+ antiporter activity in OKP cells. Dexamethasone produced a 24% stimulation in Na+/H+ antiporter activity at 10(-9) M and an approximately 40% stimulation of Na+/H+ antiporter activity at both 10(-8) and 10(-6) M. The effect of 10(-6) M dexamethasone was seen within 4 h of incubation and was due to an increase in maximal velocity (Vmax, 3.03 vs. 1.79 pH units/min) with no change in the affinity constant for sodium (KNa, 47.2 vs. 42.0 mM). The stimulatory effect of dexamethasone on Na+/H+ antiporter activity was blocked by cycloheximide and was not observed with 10(-8) M aldosterone. These data demonstrate a direct effect of glucocorticoids to stimulate Na+/H+ antiporter activity in OKP cells.

Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO 3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3′-triiodothyronine (T 3 ) increases NHE3 activity. T 3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T 3 stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5′-flanking region of the rat NHE3 gene was stimulated by T 3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T 3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T 3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance.Keywords proximal tubule; acid-base balance; NaCl homeostasis; development; promoter Sodium Chloride and NaHCO 3 absorption in the mammalian renal proximal tubule is affected by the thyroid status of the animal (10,11,21,22). The developmental maturation of proximal tubule NaCl and NaHCO 3 absorption is temporally preceded by an increase in circulating thyroid hormone levels (29). A significant portion of proximal tubule NaCl and NaHCO 3 transport is mediated by apical membrane Na/H exchange (24,25). Na/H exchange activity in renal cortical apical membrane vesicles is increased in hyperthyroid and decreased in hypothyroid animals (18,19). Although the effect on apical membrane Na/H exchange may be mediated in part by changes in glomerular filtration rate, systemic hemodynamic and/or neurohumoral changes (17), a direct effect of thyroid hormone on the maximum velocity (V max ) of the Na/H exchanger has been demonstrated in a cell culture model of the proximal tubule, the opossum kidney (OK) cell (30). With the identification of Na/H exchanger isoform cDNAs (reviewed in Ref. 28), specific reagents are now available to address the mechanisms of regulation of proximal tubule Na/H exchange. The predominant isoform responsible for proximal tubule apical membrane Na/H exchange is NHE3 (1, 6), and an opossum NHE3 homologue is expressed in OK cells. Most chronic biological effects of thyroid hormone are believed to be mediated via activation of gene transcription (26). However, one study in rat kidney showed that changes in the NHE3 transcript in response to thyroid hormone are not accompanied by changes in NHE3 protein abundance, raising doubts as to the role of increased NHE3 transcript in mediating the increased NHE3 activity (2). In the present study, we found that thyroid hormone administration to adult rats increases both apical membrane Na/H exchanger ...