{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T10:44:34Z","timestamp":1793357074149,"version":"4.5.0"},"reference-count":32,"publisher":"National Academy of Sciences","issue":"17","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2013,4,23]]},"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.1073\/pnas.1302063110","type":"journal-article","created":{"date-parts":[[2013,4,9]],"date-time":"2013-04-09T22:18:24Z","timestamp":1365545904000},"page":"7014-7019","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":85,"title":["Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients"],"prefix":"10.1073","volume":"110","author":[{"given":"Caroline M.","family":"Gorvin","sequence":"first","affiliation":[{"name":"Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Nuffield Department of Clinical Medicine, University 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