title: Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.
abstract: Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.
authors.affiliation: Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, United Kingdom.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Gorvin
authors.forename: Martijn J
authors.initials: MJ
authors.lastname: Wilmer
authors.forename: Sian E
authors.initials: SE
authors.lastname: Piret
authors.forename: Brian
authors.initials: B
authors.lastname: Harding
authors.forename: Lambertus P
authors.initials: LP
authors.lastname: van den Heuvel
authors.forename: Oliver
authors.initials: O
authors.lastname: Wrong
authors.forename: Parmjit S
authors.initials: PS
authors.lastname: Jat
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Lippiat
authors.forename: Elena N
authors.initials: EN
authors.lastname: Levtchenko
authors.forename: Rajesh V
authors.initials: RV
authors.lastname: Thakker
mesh_terms: D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D057973:Dent Disease / Q000235:genetics / Q000503:physiopathology*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry*; D004847:Epithelial Cells / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D018613:Microscopy, Confocal; D009154:Mutation / Q000235:genetics; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: C097503:CLC-5 chloride channel; D018118:Chloride Channels; C485187:PHluorin; C494807:VAMP2 protein, human; D050988:Vesicle-Associated Membrane Protein 2; D049452:Green Fluorescent Proteins