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Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.


ABSTRACT: Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

SUBMITTER: Zacchia M 

PROVIDER: S-EPMC6054333 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.

Zacchia Miriam M   Tian Xuefei X   Zona Enrica E   Alpern Robert J RJ   Preisig Patricia A PA  

Journal of the American Society of Nephrology : JASN 20180420 6


<b>Background</b> Urine citrate is reabsorbed exclusively along the renal proximal tubule <i>via</i> the apical Na<sup>+</sup>-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load <i>in vivo</i> and media acidification <i>in vitro</i> increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ET<sub>B</sub>) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.<b>Methods</b> We transiently transfected cultured opossum kid  ...[more]

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