Dents disease is a renal proximal tubular disorder characterised by low-molecular-weight proteinuria, glycosuria, hypercalciuria, phosphaturia, nephrolithiasis and abnormal urinary loss of other proteins which include insulin, parathyroid hormone (PTH) and vitamin D-binding protein, due to defective receptor-mediated endocytosis (RME). Mutations in CLC-5 cause Dents disease-1 whilst mutations in OCRL1 cause Dents disease-2 and the oculocerebrorenal syndrome of Lowe. CLC-5 is a chloride/proton antiporter which, in conjunction with the proton V-ATPase, allows progressive ionic gradients to be established in endocytic vesicles ensuring their continuation through the endosome-lysosome pathway. Studies of this pathway and Dents disease-1 would be greatly facilitated by the availability of human renal proximal tubular cell-lines from patients with Dents disease-1. We have therefore established such cell-lines, which are human conditionally-immortalised proximal tubule epithelial cell-lines (ciPTECs) harbouring one of three CLC-5 mutations, an in-frame histidine insertion at codon 30 (30:insHis), a deletion of codons 132 to 241 (del132241) and a nonsense mutation Arg637Stop, from the urine of patients with Dents disease-1. We have used these ciPTECs and a control ciPTEC to study endosomal acidification by utilising live-cell imaging of pHluorin-VAMP2, a pH-sensitive-GFP construct. Endosomal pH in a control ciPTEC was 6.01±0.17 and the pH in endosomes from the ciPTEC with the 30:insHis was similar (6.13±0.21). However in ciPTECs with the del132-241 and Arg637Stop mutations the endosomal pH was significantly more alkaline (6.63±0.17 and 7.28±0.17, respectively, P<0.05) indicating an endosomal acidification defect. Addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, thereby 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 demonstrate that despite the shared Dents disease-1 phenotype, endosomal acidification is not affected similarly by CLC-5 mutations, thereby suggesting the existence of other mechanisms in Dents disease-1.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.