α-Ketoglutarate-related inhibitors of HIF prolyl hydroxylases are substrates of renal organic anion transporters 1 (OAT1) and 4 (OAT4)

Yohannes Hagos,Johannes Schödel,Wolfgang Krick,Carsten Willam,Birgitta C. Burckhardt,Gerhard Burckhardt,Gunnar Schley

doi:10.1007/s00424-012-1140-9

Abstract

2-Oxoglutarate or α-ketoglutarate (αKG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1α (HIF-1α) in the presence of oxygen. αKG analogs are applied to stabilize HIF-1α even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of αKG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these αKG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1α revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1α. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.

Highlights

Hypoxia-inducible factors (HIFs) orchestrate the genomic response to hypoxia in a variety of cells
hypoxia-inducible factor 1α (HIF-1α) is hydroxylated at prolines 564 and 402 by three functional HIF prolyl hydroxylase domains (PHDs [7, 14, 15])
The C-terminus of the HIF-1α subunit is hydroxylated by the factor inhibiting HIF (FIH1) in the presence of oxygen which blocks interaction of HIF with the transcriptional coactivator p300 and may serve as a final transcriptional control of HIF target genes under normoxic conditions [18]

Summary

Introduction

Hypoxia-inducible factors (HIFs) orchestrate the genomic response to hypoxia in a variety of cells. HIF-1α is hydroxylated at prolines 564 and 402 by three functional HIF prolyl hydroxylase domains (PHDs [7, 14, 15]). Hydroxylation of these prolines mediates recognition of HIF by the von Hippel–Lindau tumor suppressor protein as part of an E3 ubiquitin ligase, resulting in ubiquitylation of HIF-1α and its degradation in the proteasome [19]. The C-terminus of the HIF-1α subunit is hydroxylated by the factor inhibiting HIF (FIH1) in the presence of oxygen which blocks interaction of HIF with the transcriptional coactivator p300 and may serve as a final transcriptional control of HIF target genes under normoxic conditions [18]. Under hypoxia, the dioxygen-dependent hydroxylases are not active, and the unmodified HIF-1α proteins become transcriptionally active by heterodimerization with HIF-ß

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