Molecular Mechanism of Renal Tubule Transport: α-Ketoglutarate stimulates pendrin-dependent Cl− absorption in the mouse CCD through protein kinase C

摘要

α-Ketoglutarate (α-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 Gq protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl⁻/HCO3− exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl⁻ absorption, we examined α-KG-stimulated Cl⁻ absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl⁻ absorption in CCDs from wild-type mice but had no effect on Cl⁻ absorption in pendrin knockout mice. Because Gq protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl⁻ absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl⁻ absorption. Like α-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), Ca²⁺ chelators (BAPTA, 10–20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl⁻ absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl⁻ absorption. However, in SPAK null CCDs, Cl⁻ absorption was not activated further by luminal α-KG application nor was Cl⁻ absorption reduced with the PKC inhibitor . Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl⁻/HCO3− exchange through a mechanism dependent on PKC and Ca²⁺ that involves PKC-α and PKC-δ.