1. Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients
- Author
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O Wrong, Sian E. Piret, Jonathan D. Lippiat, L.P.W.J. van den Heuvel, Caroline M Gorvin, Elena Levtchenko, Rajesh V. Thakker, Parmjit S. Jat, Brian Harding, and Martijn J. Wilmer
- Subjects
Genomic disorders and inherited multi-system disorders Energy and redox metabolism [IGMD 3] ,Endosome ,Vesicle-Associated Membrane Protein 2 ,Endocytic cycle ,Green Fluorescent Proteins ,Dent Disease ,Endosomes ,Biology ,Endocytosis ,Renal disorder Energy and redox metabolism [IGMD 9] ,Cell Line ,Kidney Tubules, Proximal ,Chloride Channels ,Humans ,Renal disorder [IGMD 9] ,Multidisciplinary ,Microscopy, Confocal ,Reabsorption ,Mitochondrial medicine Energy and redox metabolism [IGMD 8] ,Epithelial Cells ,Receptor-mediated endocytosis ,Hydrogen-Ion Concentration ,Biological Sciences ,Cubilin ,Cell biology ,Renal disorder Membrane transport and intracellular motility [IGMD 9] ,Membrane transport and intracellular motility Renal disorder [NCMLS 5] ,Mutation ,Chloride channel - Abstract
Contains fulltext : 117540.pdf (Publisher’s version ) (Closed access) 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.
- Published
- 2016