Wang Qi, Lu Ye, Yuan Min, Darling Inger M, Repasky Elizabeth A, Morris Marilyn E
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York 14260, USA.
Mol Pharm. 2006 Nov-Dec;3(6):675-85. doi: 10.1021/mp060037b.
The objectives of this study were to characterize the expression and function of monocarboxylate transporters (MCTs) in human kidney HK-2 cells and to compare the expression of MCTs in HK-2 cells to that found in human kidney. mRNA and protein expression of MCTs were determined by RT-PCR and Western analyses, respectively, while immunofluorescence staining was used to determine the membrane localization of MCT1. The driving force, transport kinetics, and inhibition of two MCT substrates, D-lactate and butyrate, were characterized in HK-2 cells. mRNA of MCT1, -2, -3, -4 isoforms were present in HK-2 cells and in human kidney cortex. MCT1 was present predominantly on the basal membranes of HK-2 cells. The cellular uptake of D-lactate and butyrate exhibited pH- and concentration-dependence (D-lactate, Km of 26.5 +/- 2.2 mM and Vmax of 72.0 +/- 14.5 nmol mg-1 min-1; butyrate, Km of 0.8 +/- 0.3 mM, Vmax of 29.3 +/- 2.5 nmol mg-1 min-1, and a diffusional clearance of 2.1 microL mg-1 min-1). The uptake of D-lactate and butyrate by HK-2 cells was inhibited by MCT analogues and the classical MCT inhibitors alpha-cyano-4-hydroxycinnamate, pCMB, and phloretin. The uptake of D-lactate and butyrate by HK-2 cells significantly decreased after transfection with small-interference RNA for MCT1. In summary, MCTs were present in both HK-2 cells and human kidney cortex, and HK-2 cells exhibited polarized MCT expression and pH-dependent transport of D-lactate and butyrate. Our results also support the usefulness of HK-2 cells as an in vitro model for studying monocarboxylate transport in renal proximal tubule cells.
本研究的目的是表征单羧酸转运体（MCTs）在人肾HK - 2细胞中的表达及功能，并比较HK - 2细胞中MCTs的表达与人肾中的表达情况。分别通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析来测定MCTs的mRNA和蛋白质表达，同时利用免疫荧光染色来确定MCT1的膜定位。在HK - 2细胞中表征了两种MCT底物D - 乳酸和丁酸的驱动力、转运动力学及抑制情况。HK - 2细胞和人肾皮质中均存在MCT1、-2、-3、-4同工型的mRNA。MCT1主要存在于HK - 2细胞的基底膜上。D - 乳酸和丁酸的细胞摄取表现出pH和浓度依赖性（D - 乳酸，米氏常数（Km）为26.5±2.2 mM，最大反应速度（Vmax）为72.0±14.5 nmol mg-1 min-1；丁酸，Km为0.8±0.3 mM，Vmax为29.3±2.5 nmol mg-1 min-1，扩散清除率为2.1 μL mg-1 min-1）。HK - 2细胞对D - 乳酸和丁酸的摄取受到MCT类似物以及经典MCT抑制剂α - 氰基 - 4 - 羟基肉桂酸、对氯汞苯甲酸（pCMB）和根皮素的抑制。用针对MCT1的小干扰RNA转染后，HK - 2细胞对D - 乳酸和丁酸的摄取显著降低。总之，MCTs存在于HK - 2细胞和人肾皮质中，且HK - 2细胞呈现出极化的MCT表达以及对D - 乳酸和丁酸的pH依赖性转运。我们的结果还支持HK - 2细胞作为研究肾近端小管细胞中单羧酸转运的体外模型的实用性。
