Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule

Wang

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

Self Cite

Na-H exchanger NHE3 and Cl-anion exchanger CFEX (SLC26A6, PAT1) play principal roles in the reabsorption of Na and Cl in the proximal tubule of the mammalian kidney. The mechanisms by which NHE3 and CFEX are localized to and maintained in the brush border of the proximal tubule are largely unknown. To investigate the possible interaction of NHE3 and CFEX with the PDZ-domaincontaining scaffolding protein PDZK1, we performed a series of in vitro interaction assays with GST-fusion proteins and native brush border membrane proteins. These studies demonstrated that, not only were NHE3 and CFEX capable of directly interacting with PDZK1, but that this interaction was mediated through their C-terminal PDZ-interaction sites. To determine whether PDZK1 interaction is essential for brush border localization of NHE3 and CFEX in vivo, we examined the expression of NHE3 and CFEX in kidneys of wild-type and PDZK1-null mutant mice by both Western analysis and immunocytochemistry. These studies indicated that, although brush border expression of NHE3 was unaffected by the loss of PDZK1, the expression of CFEX was markedly reduced. Finally, we assayed CFEX functional activity as Cl-oxalate exchange in brush border membrane vesicles and oxalate-stimulated volume absorption in microperfused proximal tubules. Consistent with the observed decrease in CFEX protein expression, both measures of CFEX functional activity were dramatically reduced in PDZK1-null animals. In conclusion, the scaffolding protein PDZK1 is essential for the normal expression and function of Cl-anion exchanger CFEX in the proximal tubule of the mammalian kidney.NHE3 ͉ proximal tubule ͉ SLC26A6

Section: The Interaction Of Cfex With Pdzk1 Is Also Mediated Through supporting

confidence: 91%

Section: The Interaction Of Cfex With Pdzk1 Is Also Mediated Through mentioning

confidence: 84%

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Role of PDZK1 in membrane expression of renal brush border ion exchangers

Wang

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

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Journal of Biological Chemistry

“…Microperfusion experiments have indicated that physiologic concentrations of formate and oxalate markedly stimulate Cl Ϫ and fluid absorption in the rat proximal convoluted tubule (13)(14)(15)(16) and that different mechanisms of anion recycling underlie the effects of formate and oxalate (17). Whereas evidence suggests that formate-stimulated NaCl uptake across the apical membrane arises from Cl Ϫ -formate exchange in parallel with Na ϩ -H ϩ exchange and H ϩ -coupled formate entry (17,18), oxalate-induced transport appears to involve oxalate-Cl Ϫ exchange in parallel with sodium-sulfate cotransport and sulfate-oxalate exchange (17). The present findings clearly raise the possibility that the Cl Ϫ -formate, Cl Ϫ -oxalate, and oxalate-sulfate exchange activities nec- essary for proximal tubule NaCl absorption may all be mediated by Slc26a6.…”

Section: Discussionmentioning

confidence: 99%

Specificity of Anion Exchange Mediated by Mouse Slc26a6

Jiang

Grichtchenko

Boron

et al. 2002

Self Cite

143

149

Recently, CFEX, the mouse orthologue of human SLC26A6, was localized to the brush border membrane of proximal tubule cells and was demonstrated to mediate Cl ؊ -formate exchange when expressed in Xenopus oocytes. The purpose of the present study was to examine whether mouse Slc26a6 can mediate one or more of the additional anion exchange processes observed to take place across the apical membrane of proximal tubule cells. The majority of Na ϩ , Cl Ϫ , and HCO 3 Ϫ filtered by the kidney is reabsorbed in the proximal tubule. Studies using isolated brush border vesicles and perfused tubules are consistent with the concept that a major fraction of Cl Ϫ entry across the apical membrane of proximal tubule cells occurs via Cl Ϫ -formate exchange and Cl Ϫ -oxalate exchange (1). The molecular identification of the transporter(s) responsible for these anion exchange activities has yet to be established with certainty. Recently, CFEX, the mouse orthologue of human SLC26A6 (2, 3), was demonstrated to be capable of mediating Cl Ϫ -formate exchange when expressed in Xenopus oocytes, and it was localized to the brush border membrane of proximal tubule cells by immunocytochemistry (4). Thus, Slc26a6 was proposed as a candidate to mediate Cl Ϫ -formate exchange in the proximal tubule (4).The purpose of the present study was to characterize the anion specificity of Slc26a6 in more detail and to test specifically the ability of Slc26a6 to mediate additional anion exchange processes known to take place across the apical membrane of proximal tubule cells, such as Cl Ϫ -oxalate exchange. We find that mouse Slc26a6 can mediate transport of oxalate, sulfate, and HCO 3 Ϫ in addition to Cl Ϫ and formate and can function in multiple exchange modes involving pairs of these anions, including Cl Ϫ -oxalate exchange. MATERIALS AND METHODS Measurements of Radiolabeled SoluteFluxes-Mouse Slc26a6 (CFEX) cDNA was subcloned into the Xenopus expression plasmid pGH19 (5) and heterologously expressed in Xenopus oocytes as described previously (4). For the experiments in this study, oocytes were injected with 50 nl of water (control) or Slc26a6 cRNA (25 ng), and transport was assayed 2 days later. For measurements of solute influx, oocytes were washed twice in 1 ml chloride-free buffer (98 mM potassium-gluconate/1.8 mM hemi-calcium-gluconate/1 mM hemi-magnesiumgluconate/5 mM Tris-Hepes, pH 7.5) and then incubated in 500 l of uptake solution (100 mM potassium-gluconate/5 mM Tris, pH adjusted to 7.5 with Hepes) containing the radiolabeled solutes to be tested ( 1 After 30-min incubation at room temperature, external isotope was removed by washing the oocytes three times with 1 ml of ice-cold chloride-free buffer. For efflux measurements, oocytes were first preloaded with radioisotope by incubating for 60 min in chloride-free buffer containing [ 14 C]oxalate. After three washes in ice-cold chloride-free buffer, the radioisotope content of oocytes was measured both initially and 15 min after suspension in solution containing test anions. Net efflux was ca...

American Journal of Physiology-Renal Physiology

“…Of the seven isoforms known to date, NHE3 is the only Na ϩ /H ϩ exchanger isoform definitively shown to be expressed in the brush-border membrane of the renal proximal tubule, on the basis of antigenic (6,13) and functional data (20,62,67). NHE3 mediates proximal tubule transcellular NaCl absorption via coupled transport with Cl Ϫ /base exchange (8,63) as well as paracellular NaCl transport by lowering luminal HCO 3 Ϫ concentration and elevating luminal Cl Ϫ concentration (52). The importance of NHE3 in sustaining extracellular fluid volume is evident by the hypovolemia and hypotension seen in NHE3 null mice (55).…”

mentioning

confidence: 99%

Insulin activates Na⁺/H⁺exchanger 3: biphasic response and glucocorticoid dependence

Klisic

Hu²,

Nief

et al. 2002

163

123

Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension. One of the target proteins of insulin action in the kidney is Na+/H+ exchanger 3 (NHE3), a principal Na+ transporter responsible for salt absorption in the mammalian proximal tubule. The molecular mechanisms involved in activation of NHE3 by insulin have not been studied so far. In opossum kidney (OK) cells, insulin increased Na+/H+exchange activity in a time- and concentration-dependent manner. This effect is due to activation of NHE3 as it persisted after pharmacological inhibition of NHE1 and NHE2. In the early phase of stimulation (2–12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, enhanced NHE3 activity was accompanied by an increase in total and cell surface NHE3 protein and NHE3 mRNA abundance. All the effects of insulin on NHE3 activity, protein, and mRNA were amplified in the presence of hydrocortisone. These results suggest that insulin stimulates renal tubular NHE3 activity via a biphasic mechanism involving posttranslational factors and an increase in NHE3 gene expression and the effects are dependent on the permissive action of hydrocortisone.