Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

Abstract

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. Receptormediated 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.

Renal Handling of Circulating and Renal-Synthesized Hepcidin and Its Protective Effects against Hemoglobin–Mediated Kidney Injury

“…To test this hypothesis, we exploited two in vitro models. Using human conditionally immortalized proximal tubular epithelial cells (ciPTECs) 30 that express megalin, 31 we showed intracellular uptake of both hhep25 (20 nM) and Alexa546-labeled hemoglobin (Alexa-Hb; 5 nM) during 1 hour of incubation ( Figure 7A). Incubation of ciPTECs with both compounds simultaneously for 1 hour resulted in significantly less uptake of both (P,0.001) compared with incubation with either one alone ( Figure 7B), which indicates competition for uptake between hhep25 and hemoglobin.…”

ClC-5: Physiological role and biophysical mechanisms

“…Surprisingly, in a study performed in transfected HEK cells, Smith et al observed that the mutants G57V and R280P showed reduced surface expression and current compared to WT but did not alter (G57V) and even enhanced (R280P) endosomal acidification [37]. Recently, Gorvin et al investigated the process of endocytosis in conditionally immortalized proximal-tubular epithelial cell lines (ciPTEC) from patients with Dent's disease due to mutations in ClC-5 [39]. With this approach it was shown that the insertion of a His residue at position 30 (30:insH) reduces receptor mediated endocytosis but does not change endosomal pH [39].…”

“…Recently, Gorvin et al investigated the process of endocytosis in conditionally immortalized proximal-tubular epithelial cell lines (ciPTEC) from patients with Dent's disease due to mutations in ClC-5 [39]. With this approach it was shown that the insertion of a His residue at position 30 (30:insH) reduces receptor mediated endocytosis but does not change endosomal pH [39]. These studies indicate that the mechanism by which ClC-5 mutations lead to Dent's disease is not necessarily linked to defective endosomal acidification, demanding the revision of a well-established paradigm.…”

Dentova bolezen

“…Gen, ki je odgovoren za motnjo kloridnega kanalčka Cl-/H+ transporterja (CLC-5), se nahaja na kromosomu Xp11.22 in kodira lizosomski transportni protein CLC-5. Lociran je skupaj z ATP-protonsko črpal-ko na endosomu proksimalnih tubulnih celic (2,4,12). Klinično se v večini primerov kaže kot zelo težka ali blaga oblika s hiperkalciurijo, bolezensko proteinurijo (prevladovanje beljakovin z majhno molekulsko maso), nefrokalcinozo in kronično ledvično boleznijo (1,5).…”