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

Abstract

<b>Background</b> Urine citrate is reabsorbed exclusively along the renal proximal tubule <i>via</i> the apical Na⁺-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_B) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.<b>Methods</b> We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ET_B 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.<b>Results</b> Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 <i>in vitro</i> 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/ET_B stimulation of NaDC-1 <i>in vitro</i> 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.<b>Conclusions</b> 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/ET_B in this process.