Receptor-mediated endocytosis (RME), involving megalin and cubilin, mediates renal proximal-tubular reabsorption of glucose, proteins and hormones including insulin, parathyroid-hormone and vitamin D. RME disruption occurs in Dents disease patients with mutations of the chloride/proton antiporter, CLC-5, who suffer from low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis and renal failure. To further investigate the RME role of CLC-5 we established conditionally immortalized proximal-tubular epithelial cell-lines (ciPTECs) from three patients with CLC-5 mutations (30insHis, Arg637Stop and del132-241). Clonal populations of urinary cells were established by selection of aminopeptidase-N-positive cells using fluorescence-activated cell sorting, and characterised by flow cytometry, RT-PCR, western blot and confocal microscopy to establish expression of ATP-binding cassette sub-family C member 4 (ABCC4), Aquaporin-1, dipeptidyl peptidase-4 (DPPIV/CD26) and P-glycoprotein to demonstrate their proximal-tubular origin. Endocytosis was investigated by fluorescent-labelled albumin and transferrin uptake assays with and without 10-fold excess of unlabelled albumin or transferrin, and compared to two control ciPTECs. Confocal microscopy using the tight junction marker ZO-1, and EB1 which is specific for the plus-end of microtubules, were used to demonstrate that the ciPTECs polarised. Fluorescent-albumin-uptake in the three Dents disease ciPTECs was significantly decreased to 2065% of control values (P<0.02). In ciPTECs with the 30insHis and Arg637Stop, albumin-uptake was demonstrated to be further reduced by competition with unlabelled albumin and transferrin (13.23±3.01% and 16.44±7.43%, respectively for 30insHis (P<0.02); and 36.16±6.62% and 43.72±3.54%, respectively for Arg637Stop (P<0.05) when compared to uptake without competition), thereby confirming the specificity of the albumin uptake by the megalin-cubilin RME pathway in these ciPTECs. Furthermore, transferrin-uptake was similarly reduced in the ciPTECs with CLC-5 mutations, consistent with impairment of CLC-5-megalin-cubilin RME in these Dents disease ciPTECs. Thus, these Dents disease ciPTECs which have defective RME, will help in elucidating the mechanisms involved in renal proximal-tubular reabsorption of solutes.