Qi Wang1, Ye Lu, Marilyn E Morris. 1. Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, 517 Hochstetter Hall, Amherst, New York 14260, USA.
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(-1)min(-1); GHB: K (m) of 2.07 +/- 0.79 mM, V (max) of 27.6 +/- 9.3 nmol mg(-1)min(-1), and a diffusional clearance of 0.54 +/- 0.15 microl mg(-1)min(-1)), but not sodium-dependence. alpha-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.
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(-1)min(-1); GHB: K (m) of 2.07 +/- 0.79 mM, V (max) of 27.6 +/- 9.3 nmol mg(-1)min(-1), and a diffusional clearance of 0.54 +/- 0.15 microl mg(-1)min(-1)), but not sodium-dependence. alpha-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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