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1 Department of Physiology and Pathophysiology, Faculty of Medicine, University of Witten/Herdecke, Witten, Germany
2 Department of Cell Biology, Aarhus University, Aarhus, Denmark; Department of Biochemistry, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland
3 Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom
4 Department of Cell Biology, Aarhus University, Aarhus, Denmark
* To whom correspondence should be addressed. E-mail: craig.smith{at}manchester.ac.uk.
The H+ coupled poly-ligand 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 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 Cl.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 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially co-localized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labelling, 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 WKPT-0293 Cl.2 cells. Within the cells, Alexa Fluor® 546-transferrin co-localized 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+.
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