Wang Qi, Lu Ye, Morris Marilyn E
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.
Pharm Res. 2007 Jun;24(6):1067-78. doi: 10.1007/s11095-006-9228-6. Epub 2007 Mar 22.
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.
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.
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.
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.
我们实验室之前的研究表明，γ-羟基丁酸（GHB）可能通过单羧酸转运体（MCT）介导进行肾脏重吸收。本研究的目的是利用HK-2细胞表征GHB的肾脏转运以及MCT在GHB肾脏转运中的作用。
采用蛋白质免疫印迹法检测MCT1、2和4的蛋白表达。进行细胞摄取和定向通量研究以探究GHB和L-乳酸的转运。采用RNA干扰试验研究MCT亚型在GHB转运中的作用。
HK-2细胞中存在MCT1、2和4。L-乳酸和GHB的细胞摄取表现出pH和浓度依赖性（L-乳酸：米氏常数（K(m)）为6.5±1.1 mM，最大反应速度（V(max)）为340±60 nmol mg(-1)min(-1)；GHB：K(m)为2.07±0.79 mM，V(max)为27.6±9.3 nmol mg(-1)min(-1)，扩散清除率为0.54±0.15 μl mg(-1)min(-1)），但不依赖于钠。α-氰基-4-羟基肉桂酸（CHC）竞争性抑制GHB和L-乳酸的摄取，抑制常数（K(i)）分别为0.28±0.1 mM和0.19±0.03 mM。使用针对MCT1的小干扰RNA（siRNA），MCT1的蛋白表达以及L-乳酸和GHB的摄取均显著降低。HK-2细胞中对MCT2进行siRNA处理可使GHB摄取降低17%，对MCT4进行siRNA处理未显示对GHB摄取有抑制作用。在pH 6.0至pH 7.4的pH梯度存在下，GHB呈现出从顶侧室到基底室穿过HK-2单层的定向通量。
这些数据表明，MCT1是肾小管细胞中GHB转运的重要转运体，负责肾脏中GHB的重吸收。
