α-Ketoglutarate stimulates pendrin-dependent Cl~- absorption in the mouse CCD through protein kinase C

摘要

α-Ketoglutarate (a-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a G_q, protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), C1~-/(HCO_3)~- exchange increases upon a-KG binding to the OXGR1. To determine the signaling pathway(s) by which a-KG stimulates Cl absorption, we examined a-KG-stimulated Cl absorption in isolated perfused mouse CCDs. a-KG increased electro-neutral Cl~- absorption in CCDs from wild-type mice but had no effect on Cl~- absorption in pendrin knockout mice. Because G_q protein-coupled receptors activate PKC, we hypothesized that a-KG stimulates Cl ~ absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, a-KG-stimulated Cl absorption. Like a-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl~- absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca~(2+) chelators (BAPTA, 10-20 (μM), or PKC-a or -8 gene ablation eliminated a-KG-stimulated Cl absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary a-KG excretion, renal pendrin abundance, and CCD Cl" absorption. However, in SPAK null CCDs, Cl~- absorption was not activated further by luminal a-KG application nor was Cl" absorption reduced with the PKC inhibitor GF109203. Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, a-KG stimulates pendrin-dependent Cl~-/HCO_3~- exchange through a mechanism dependent on PKC and Ca~(2+) that involves PKC-a and PKC-8.