CIC-5: A chloride channel with multiple roles in renal tubular albumin uptake

CIC-5 is a chloride (Cl-) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in CIC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in CIC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. CIC-5 is typically regarded as an intracellular Cl- channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. CIC-5 was postulated to play a key role in transporting the Cl- ions required to compensate for the movement of H+ during endosomal acidification. However, more recent studies suggest additional roles for CIC-5 in the endocytosis of albumin. CIC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in CIC-5 affect the trafficking of v-H+-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of CIC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule. (c) 2005 Elsevier Ltd. All rights reserved.

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds - Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney ( OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.