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Monocarboxylate Transporter (MCT) Mediates the Transport of γ-Hydroxybutyrate in Human Kidney HK-2 cells

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Abstract

Purpose

Previous studies in our laboratory have suggested that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCT). The objectives of this study were to characterize the renal transport of GHB using HK-2 cells and the role of MCT in the renal transport of GHB.

Materials and Methods

Western blot was used to detect the protein expression of MCT1, 2, and 4. Cellular uptake and directional flux studies were conducted to investigate the transport of GHB and L-lactate. RNA interference assay was used to investigate the involvement of MCT isoforms in the transport of GHB.

Results

MCT1, 2 and 4 were present in HK-2 cells. The cellular uptake of L-lactate and GHB exhibited pH- and concentration-dependence (L-lactate: K m of 6.5 ± 1.1 mM and V max of 340 ± 60 nmol mg−1min−1; GHB: K m of 2.07 ± 0.79 mM, V max of 27.6 ± 9.3 nmol mg−1min−1, and a diffusional clearance of 0.54 ± 0.15 μl mg−1min−1), but not sodium-dependence. α-Cyano-4-hydroxycinnamate (CHC) competitively inhibited the uptake of GHB and L-lactate with inhibition constants (K i) of 0.28 ± 0.1 mM, and 0.19 ± 0.03 mM, respectively. Using small-interference RNA (siRNA) for MCT1, the protein expression of MCT1 and the uptake of L-lactate and GHB were significantly decreased. The siRNA treatment of MCT2 in HK-2 cells inhibited the uptake of GHB by 17%, and the siRNA treatment of MCT4 demonstrated no inhibition of GHB uptake. GHB exhibited a directional flux across HK-2 monolayer from apical to basal chambers in the presence of a pH gradient of pH 6.0 to pH 7.4.

Conclusion

These data suggest that MCT1 represents an important transporter for GHB transport in renal tubule cells, responsible for the reabsorption of GHB in the kidney.

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Abbreviations

CHC:

α-cyano-4-hydroxycinnamate

DIDS:

4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid

GHB:

γ-hydroxybutyrate

MCT:

monocarboxylate transporter

PCMB:

p-chloro-mercuribenzoic acid

TEA:

tetraethylammonium chloride

SiRNA:

small interference RNA

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Acknowledgments

Support was provided by NIH grant DA14988, by a grant from the Western New York Kidney Foundation/Upstate New York Transplant Service and by a Mark-Diamond grant from University at Buffalo.

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Correspondence to Marilyn E. Morris.

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Wang, Q., Lu, Y. & Morris, M.E. Monocarboxylate Transporter (MCT) Mediates the Transport of γ-Hydroxybutyrate in Human Kidney HK-2 cells. Pharm Res 24, 1067–1078 (2007). https://doi.org/10.1007/s11095-006-9228-6

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