Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes:: implications for renal handling of protein-metal complexes
Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes:: implications for renal handling of protein-metal complexes
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DOI：
10.1152/ajprenal.00359.2005
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发表时间：
2006-06-01
影响因子：
4.2
通讯作者：
Smith, CP
中科院分区：
文献类型：
--
作者：
Abouhamed, M;Gburek, J;Smith, CP
H+偶联多配体转运蛋白二价金属转运蛋白1（DMT 1）在哺乳动物铁稳态中起着关键作用。它具有广泛的表达模式，包括与铁获取和储存相关的组织。有趣的是，它在肾脏中也高度表达，但其在该组织中的功能尚不清楚。本研究的目的是确定DMT 1在近端小管细胞中的细胞位置，作为确定该蛋白在肾脏中作用的第一步。为此，我们使用大鼠肾脏和S1近端小管衍生的WKPT-0293 C1.2细胞系进行了RT-PCR和免疫染色实验。RT-PCR显示，编码所有四个DMT 1剪接变体的mRNA存在于从大鼠肾皮质或WKPT-0293 C1.2细胞提取的RNA中。大鼠肾皮质或WKPT-0293 C1.2细胞的免疫染色显示，DMT 1蛋白在细胞内表达，而不存在于质膜。DMT 1的表达与晚期内体/溶酶体蛋白LAMP 1和组织蛋白酶-L部分共定位。使用免疫金标记，显示DMT 1在晚期内体/溶酶体的膜中表达。仅在应用于WKPT 0293 C1.2细胞的顶膜后观察到Alexa Fluor 546-转铁蛋白的摄取。在这些细胞内，Alexa Fluor 546-转铁蛋白与DMT 1共定位。总之，肾近端小管细胞表达DMT 1细胞器的膜，包括晚期内体/溶酶体，与顶部隔离的转铁蛋白的加工。这些发现对肾铁处理和可能对也是DMT 1配体的肾毒性金属（包括Cd 2+）的处理具有影响。
The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 C1.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 C1.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 C1.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT0293 C1.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.