TY - JOUR
T1 - Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients
AU - Gorvin, Caroline M.
AU - Wilmer, Martijn J.
AU - Piret, Sian E.
AU - Harding, Brian
AU - Van Den Heuvel, Lambertus P.
AU - Wrong, Oliver
AU - Jat, Parmjit S.
AU - Lippiat, Jonathan D.
AU - Levtchenko, Elena N.
AU - Thakker, Rajesh V.
PY - 2013/4/23
Y1 - 2013/4/23
N2 - Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease becauseofmutationsofthechloride/protonantiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight pro-teinuria, 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 im-agingofpHluorin-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 additionof 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, ourstudies, 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 endocy-tosis that may not be coupled.
AB - Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease becauseofmutationsofthechloride/protonantiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight pro-teinuria, 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 im-agingofpHluorin-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 additionof 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, ourstudies, 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 endocy-tosis that may not be coupled.
UR - http://www.scopus.com/inward/record.url?scp=84876855574&partnerID=8YFLogxK
U2 - 10.1073/pnas.1302063110
DO - 10.1073/pnas.1302063110
M3 - Article
C2 - 23572577
AN - SCOPUS:84876855574
SN - 0027-8424
VL - 110
SP - 7014
EP - 7019
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 17
ER -
