1999 Oxford University Press 247–257Human Molecular Genetics, 1999, Vol. 8, No. 2

Intra-renal and subcellular distribution of the humanchloride channel, CLC-5, reveals a pathophysiologicalbasis for Dent’s disease Olivier Devuyst 1, Paul T. Christie 2, Pierre J. Courtoy 3, Renaud Beauwens 4 and Rajesh V. Th akker 2,*

1Division of Nephrology and 3Cell Unit, Christian de Duve Institute of Cellular Pathology, University of LouvainMedical School, B-1200 Brussels, Belgium, 2MRC Molecular Endocrinology Group, MRC Clinical Sciences Centre,Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK and 4Department ofPhysiopathology, Free University of Brussels Medical School, B-1070 Brussels, Belgium

Received August 7, 1998; Revised and Accepted November 5, 1998

Dent’s disease, which is a renal tubular disordercharacterized by low molecular weight proteinuria,hypercalciuria and nephrolithiasis, is associated withinactivating mutations of the X-linked chloride channel,CLC-5. However, the manner in which a functional lossof CLC-5 leads to such diverse renal abnormalitiesremains to be defined. In order to elucidate this, weperformed studies to determine the segmental express-ion of CLC-5 in the human kidney and to define its intra-cellular distribution. We raised and characterizedantisera against human CLC-5, and identified byimmunoblotting an 83 kDa band corresponding toCLC-5 in human kidney cortex and medulla. Immuno-histochemistry revealed CLC-5 expression in theepithelial cells lining the proximal tubules and the thickascending limbs of Henle’s loop, and in intercalatedcells of the collecting ducts. Studies of subcellularhuman kidney fractions established that CLC-5distribution was associated best with that of Rab4,which is a marker of recycling early endosomes. Inaddition, confocal microscopy studies using theproximal tubular cell model of opossum kidney cells,which endogenously expressed CLC-5, revealed thatCLC-5 co-localized with the albumin-containing endo-cytic vesicles that form part of the receptor-mediatedendocytic pathway. Thus, CLC-5 is expressed at multiplesites in the human nephron and is likely to have a rolein the receptor-mediated endocytic pathway. Further-more, the functional loss of CLC-5 in the proximaltubules and the thick ascending limbs provides anexplanation for the occurrences of low molecularweight proteinuria and hypercalciuria, respectively.These results help to elucidate further the patho-

physiological basis of the renal tubular defects ofDent’s disease.

INTRODUCTION

Mutations of the renal-specific chloride channel (CLCN5) gene,which is located on chromosome Xp11.22, result in Dent’sdisease, which is a renal tubular disorder characterized by lowmolecular weight (<40 kDa) proteinuria (e.g. α1 and β2microglobins, lysozyme and retinol-binding protein), albuminuria,hypercalciuria, nephrocalcinosis, nephrolithiasis and renal failure(1–4). In addition, other renal proximal tubular defects, whichinclude aminoaciduria, phosphaturia, glycosuria, kaliuresis,uricosuria and an impairment of urinary acidification may alsooccur (1). CLCN5, which in man is expressed predominantly inthe kidney, belongs to the family of mammalian voltage-gatedchloride channel genes (CLCN1–CLCN7, CLCNKa andCLCNKb) that encode proteins (CLC-1–CLC-7, CLC-Ka andCLC-Kb) with ∼12 transmembrane domains (Fig. 1) (5–7). Thesechloride channels are important for the control of membraneexcitability, transepithelial transport and possibly regulation ofcell volume. To date, only mutations of CLCN1, CLCNKb andCLCN5 have been reported to be associated with humandisorders, and these are the myotonia disorders of Thomsen andBecker (8), a form of Bartter’s syndrome (9) and Dent’s disease(2,3), respectively (7). Heterologous expression of wild-typeCLC-5 in Xenopus oocytes results in chloride conductance (2)which is markedly reduced or abolished by the CLC-5 mutationsof Dent’s disease (2,3). However, the mechanisms whereby a lossof this chloride conductance leads to hypercalciuria and theproximal renal tubular defects, e.g. low molecular weightproteinuria, remain to be elucidated.

Calcium reabsorption occurs in a large segment of the nephronthat includes the proximal tubule, the thick ascending limb ofHenle’s loop and the distal tubule (10), whilst that of albumin andlow molecular weight proteins is restricted to the proximal tubuleand involves endocytosis with subsequent transport to the acidic

*To whom correspondence should be addressed. Tel: +44 181 383 3014; Fax: +44 181 383 8306; Email: rthakker@rpms.ac.uk

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Figure 1. Characterization of human CLC-5 antisera. (A) Schematic representation of the topology of CLC-5. The correct topology of CLC-5, which consists of 746amino acids (2,6), is unknown, and the predicted topology of the CLC-5 putative transmembrane domains (D1–D13) is based upon a model previously described (2).Rabbit polyclonal antisera were raised against the two CLC-5 epitopes (asterisks) whose locations between D1 and D2 (L1-2), and D8 and D9 (L8-9), respectively,are shown. The position of the mutation W279X which has been observed three times in Dent’s disease patients (2,3) and which results in a truncated (30 kDa) channelis also shown. (B) Immunoblotting of in vitro translated CLC-5 products. Control protein luciferase (61 kDa, lanes 1, 10% gel), wild-type CLC-5 protein (83 kDa,lanes 2, 10% gel) and W279X mutant CLC-5 protein (30 kDa, lanes 3, 14% gel), produced by using in vitro translation, were loaded (20 µg of protein) in each lane.Control [35S]methionine incorporated in vitro translation products are shown (35S), with the molecular mass standards indicated on the left. Western blots using twodifferent rabbit anti-CLC-5 sera (L8-9 and L1-2) were developed as described (46), and the molecular mass standards are indicated in kDa on the left for the 10% gelsand on the right for the 14% gels. Antiserum L1-2 detected both the wild-type 83 kDa and mutant 30 kDa CLC-5 products, while antiserum L8-9 detected only thewild-type 83 kDa CLC-5 product, as predicted from a consideration of the topology (A). (C) Immunoblotting of CLC-5 peptides (residues 108–125 and 382–399)and corresponding CLC-3 and CLC-4 peptides [250 ng (lane 1), 100 ng (lane 2) and 20 ng (lane 3)]. The antisera L1-2 and L8-9 specifically revealed immunoreactivityto the respective CLC-5 peptides and not to the CLC-3 or CLC-4 peptides. The pre-immune sera showed no immunoreactivity to these peptides (data not shown).(D) Tissue expression of CLC-5. Thirty micrograms of total protein extracts obtained from several human tissues were loaded on a 7.5% gel that was transferred andprobed either with the anti-CLC-5 L1-2 or L8-9 (1:1000 dilution). Two major bands at ∼83 kDa (arrow) and 57 kDa were detected. The 57 kDa band, which was detectedby the pre-immune sera (Fig. 2A) and control IgG (data not shown) in some tissue extracts, was found to be non-specific for CLC-5. However, the 83 kDa band, whichwas specific (Fig. 2A) and corresponded to CLC-5, was expressed predominantly in the kidney (lane 5) and to a lesser extent in placenta (lane 7) and pancreas (lane 1).CLC-5 was not detected in extracts from liver (lane 2), brain (hippocampus, lane 3; olfactory cortex, lane 4) and skeletal muscle (lane 6).

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vacuolar–lysosomal system (11). A loss of CLC-5 function maydecrease chloride influx in the endocytic vesicles and therebyprevent the dissipation of the charge that is generated by theelectrogenic H+-ATPase pump for the provision of the acidicenvironment (6,11). This would lead potentially to impairedendosomal acidification and inefficient reabsorption of the lowmolecular weight proteins (11,12). In addition, a decreasedreabsorption of chloride ions in the proximal tubule may alsoresult in reduced calcium reabsorption (13). The identification ofthe specific segments of the nephron that express CLCN5 wouldhelp to clarify these and other mechanisms. Such studies havebeen performed in the rat kidney (14–16). Thus, in situhybridization revealed CLCN5 expression consistently in the typeα intercalated cells of the rat collecting duct but only occasionallyin the proximal tubules (14). However, the use of RT–PCR on ratmicro-dissected tubules revealed CLCN5 expression in all of thesegments of the nephron, although expression in the glomerulusand the S2 segment of the proximal tubule was low (15). Morerecently, immunohistochemistry studies of rat kidney havelocalized CLC-5 expression to the proximal tubular cells and toboth types of intercalated cells in the collecting duct (16). Thesedifferent findings (14–16) in the rat nephron do not explain all theabnormalities observed in patients with Dent’s disease. In addition,any extrapolation of these findings from the rat to the human kidneyrequires caution, as there are significant developmental, functionaland morphological differences between the two mammaliankidneys (17–19). Moreover, the tissue expression of CLCN5 inthe two mammals also differs, with CLCN5 being expressed in therat kidney, brain, lung and liver (15), but being expressedpredominantly only in the human kidney (6).

In order to determine the segmental expression of CLC-5 inhuman kidney and to define its intracellular localization, weraised and characterized polyclonal antisera to two epitopes ofCLC-5 (Fig. 1) and used them for expression studies using normalhuman kidney sections and subcellular fractions, respectively. Inaddition, we investigated further, by confocal microscopy, theintracellular distribution and putative function of CLC-5 by useof opossum kidney (OK) cells, which represent a well-establishedmodel for proximal tubular epithelial cells (20). Our resultsrevealed that CLC-5 is localized to the endosomes and isexpressed consistently in the human proximal tubule, the thickascending limb of Henle’s loop and in the intercalated cells of thecollecting duct. Thus, CLC-5 is expressed more widely in the humanthan the rat nephron, and our observed sites of CLC-5 expressionhelp to provide further insight into the pathophysiological basis forthe renal tubular defects observed in Dent’s disease.

RESULTS

Characterization of CLC-5 antisera

Two antisera (L1-2 and L8-9) raised in rabbits against the putativeloops (Fig. 1A), between D1 and D2, and D8 and D9, respectively,of human CLC-5, were characterized against in vitro translatedwild-type CLC-5 and mutant W279X proteins (Fig. 1B).[35S]methionine incorporation (Fig. 1B) verified the efficiency ofthe in vitro translation, and western blot analysis using the L1-2antiserum detected both the truncated and wild-type CLC5proteins, whereas the L8-9 antiserum detected only the wild-typeCLC-5 protein (Fig. 1B), as expected from the putative locationsof their respective epitopes. In addition, an assessment of the

specificity of L1-2 and L8-9 antisera, using the CLC-3, CLC-4and CLC-5 peptides, together with the pre-immune and immunesera demonstrated that both antisera were specific for CLC-5(Fig. 1C). Furthermore, the human tissue expression of CLC-5,as detected by western blot analysis (Fig. 1D), was found to bepredominantly in the kidney and to a lesser extent in the placenta andpancreas; this is consistent with our previous observations of CLCN5mRNA by northern blot analysis (6). This predominantly renalexpression of CLC-5 is markedly different from that of CLC-3 andCLC-4 which are expressed at high levels in the brain (hippocampusand olfactory cortex) and skeletal muscle, respectively (21,22).Thus, the predominant renal expression of CLC-5 detected by theL1-2 and L8-9 CLC-5 antisera, and the lack of strong signals fromextracts of the other human tissues, indicates that these antiseraare specific for CLC-5 and do not have cross-reactivity to CLC-3and CLC-4. Both antisera L1-2 and L8-9, which were usedsystematically in all of the studies, yielded similar results andidentified the same structures by immunoblotting and immuno-staining. In addition, similar results were obtained using affinity-purified CLC-5 antibodies (Fig. 2A).

CLC-5 expression in the human kidney

The L8-9 anti-CLC-5 serum identified two bands at 83 and ∼57 kDain human kidney extracts (Fig. 2A). The ∼57 kDa band wasdetectable at similar densities with the pre-immune serum andwas therefore considered to be non-specific for CLC-5. However,the 83 kDa band, which corresponded to the predicted molecularmass of the 746 amino acid CLC-5, was not detected by thepre-immune serum. In addition, this 83 kDa band was detectedspecifically by both (L1-2 and L8-9) of the affinity-purifiedantibodies (Fig. 2A) and by the L1-2 antiserum (Fig. 1D). Thus,the 83 kDa band was regarded as specific for CLC-5. Acomparison of the signal intensities revealed that CLC-5 expressionwas higher in the renal cortex than in the medulla (Fig. 2A). Thespecificity for the 83 kDa band in human kidney extracts wasconfirmed further using peptide adsorption (Fig. 2B). Theintensity of the 83 kDa band (lane 1) was markedly decreasedwhen the L8-9 antiserum was pre-adsorbed with the cognatepeptide of human CLC-5 (lane 2), but not affected whenpre-adsorbed with a control peptide from human CLC-3 (lane 3)or CLC-4 (lane 4), while it was absent with the pre-immune serumL8-9 (lane 5). These results indicate that the CLC-5 antisera arespecific for the renal expressed CLC-5 83 kDa band.

The subcellular distribution of CLC-5 expression was studiedby using human kidney fractions (Fig. 2C) obtained by differentialcentrifugation (Fig. 3), as previously described for the isolationof subcellular fractions from liver and kidney (23–25). Thedistinct nature of these subcellular fractions (LSP, LSS, HSP, HSSand PM) was demonstrated by their differing protein compositions(assessed by SDS–PAGE and Coomassie staining), their differingvesicular content [assessed by electron microscopy (26)] and bytheir differing specific marker enzymes content (27). Fluid-phaseendocytosis tracers such as dextran were located in both LSP andHSP fractions, whereas receptor-mediated endocytosis tracerssuch as albumin were primarily in the HSP fraction (24).Repeated immunoblot analyses showed that the 83 kDa band wasequally abundant in the HSP and LSP, but consistently lessabundant in the PM fraction (Fig. 2C). A faint band was alsoobserved in the LSS (lane 2) upon longer exposure. A comparisonof the distribution of CLC-5 immunoreactivity in these subcellular

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Figure 2. Western blot analysis and subcellular expression of CLC-5 in thehuman kidney. (A) Expression of CLC-5 in human kidney. Tissue extracts(30 µg of total protein per lane) from cross-sections (lanes 1 and 4), medulla(lanes 2 and 5) and cortex (lanes 3 and 6–8) obtained from two human kidneyswere electrophoresed (7.5% gel) and transferred to nitrocellulose. The blotswere probed with: (i) the anti-CLC-5 L8-9 (lanes 1–3); (ii) the correspondingpre-immune serum (lanes 4–6) at the same dilution (1:1000); or (iii) theaffinity-purified L8-9 (lane 7) or L1-2 (lane 8) antibodies (1:100 dilution), andvisualized by ECL. The films were exposed for 2 min (lanes 1–6) or 10 min(lanes 7 and 8). The molecular mass standards are given in kDa. An 83 kDaband corresponding to the predicted molecular mass of human CLC-5 isidentified specifically in all extracts (arrow). The second band at ∼57 kDa isnon-specific as it is also detected with the pre-immune serum. Similar resultswere also obtained with the L1-2 antiserum. (B) The specificity of the 83 kDaband detected with the anti-CLC-5 serum L8-9 in the human kidney wasassessed by peptide adsorption. A human kidney extract (30 µg of total proteinper lane) was electrophoresed (7.5% gel) and transferred to nitrocellulose.Following demonstration of similar transfer efficacy by Ponceau red (Sigma)staining, nitrocellulose strips were cut and probed with: (i) L8-9 antiserum(lane 1); (ii) L8-9 antiserum pre-adsorbed with an excess of the specific CLC-5peptide (lane 2) or the corresponding peptide derived from CLC-3 (lane 3) orCLC-4 (lane 4); and (iii) the pre-immune serum L8-9 (lane 5). The intensity ofthe 83 kDa band (arrow) was markedly reduced by adsorption with the CLC-5peptide (lane 2) but not affected with the CLC-3 or CLC-4 peptides (lanes 3 and4). All films were exposed for 30 s. (C) Subcellular expression of CLC-5 in thehuman kidney. The five subcellular fractions of human kidney prepared bydifferential centrifugation (Fig. 3) were loaded (30 µg of total protein in eachlane) and the blot was probed with the antiserum L8-9. The 83 kDa bandcorresponding to CLC-5 was detected with similar intensity in LSP and HSPfractions (lanes 1 and 3, arrowhead), and to a lesser extent in the PM fraction(lane 5). These fractions were investigated similarly using antibodies (dilution1:200–1:500) raised against the Rab4 (14% gel), Rab5a (12% gel) and Rab6isoforms (14% gel), β-COP (7.5% gel) or vacuolar H+-ATPase (12% gel). Theexpression of Rab4, which was more abundant in the LSP and HSP than in thePM fractions, paralleled that of CLC-5, in contrast to that of β-COP which wasexpressed almost exclusively in the HSP fraction. Rab5a and H+-ATPase weremost enriched in the LSP fraction (lane 1). Representative immunoblots of atleast two different experiments are shown.

fractions with that of several markers of subcellular compartmentswas undertaken (Fig. 2C). These markers included: Rab4, whichis localized throughout the early endosome and plasma membranerecycling pathway; Rab5a which is distributed in plasmamembrane, clathrin-coated vesicles and early endosomes; Rab6,which is present in the Golgi stacks and trans-Golgi network (28);β-COP, which is a marker of small vesicles of the secretorypathway (29); and H+-ATPase, the vacuolar subunit of the protonpump, which is detected in the plasma membrane and inclathrin-coated vesicles, and is concentrated in intracellularorganelles including endosomes and lysosomes (30,31). Ouranalysis showed that the subcellular distribution of CLC-5 did notparallel that of β-COP which is a marker for the Golgi complexthat is enriched in the HSP fraction (Fig. 2C). In contrast, thedistribution of CLC-5 was similar to that of H+-ATPase but wasassociated best with that of Rab4, thus being consistent with theearly endosome and plasma membrane recycling pathway.

Immunolocalization of CLC-5 in the human kidney

Immunoreactivity for CLC-5 in the human nephron (Fig. 4) wasdetected in the cortical proximal tubules (Fig. 4A–D), themedullary thick ascending (Fig. 4E, F and H) and thin descendinglimbs (Fig. 4F) of Henle’s loop, and in intercalated cells of thecollecting ducts (Fig. 4K and L). Comparable immunoperoxidaseresults, using paraformaldehyde kidney sections, were obtained

with the L1-2 and L8-9 antisera, and with the affinity-purifiedantibodies. In addition, the use of specific antibodies facilitatedthe identification of: proximal tubules (vacuolar H+-ATPase);thin descending limbs (AQP1) and medullary thick ascendinglimbs (Tamm–Horsfall protein) of Henle’s loop; and intercalatedcells of the collecting ducts (H+-ATPase). In the proximal renaltubules, CLC-5 expression was intracellular (Fig. 4B) and lesspolarized than that of H+-ATPase (Fig. 4C). This intracellularexpression of CLC-5 was also observed in the medullary limbs ofHenle’s loop (Fig. 4E, F and H). Controls that includedpre-adsorbed antisera (Fig. 4D and J) and pre-immune sera (Fig. 4Nand O) demonstrated the specificity of the immunoreactivity.Similar results were obtained by using immunofluorescence onacetone-fixed frozen sections from two normal human kidneys(data not shown).

CLC-5 expression, and co-localization studies withendocytosis tracers in OK cells

In order to investigate further the intracellular localization ofCLC-5, studies were performed using OK cells which have beenestablished as a model for proximal tubule epithelial cells (20,32).OK cells were demonstrated by RT–PCR to express the CLCN5gene (Fig. 5A), and the presence of the 83 kDa CLC-5-specificband was demonstrated by immunoblot analysis (Fig. 5B and C).The putative endosomal localization of CLC-5 in OK cells was

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Figure 3. Subcellular fractionation of human kidney by differential centrifugation. Human kidney homogenates and subcellular fractions were prepared by differentialcentrifugation, as described (23–25). The procedure yielded five subcellular fractions (grey boxes): an LSP with corresponding supernatant (LSS), an HSP withcorresponding supernatant (HSS) and a PM fraction. These fractions were characterized by immunoblotting (Fig. 2).

investigated further by confocal microscopy examination usingdextran–fluorescein isothiocyanate (FITC)–lysine and albumin–FITC, which are two endocytic tracers that preferentially followthe fluid-phase and receptor-mediated endocytosis pathways,respectively (20,33). The time course of internalization ofalbumin–FITC (Fig. 6A–C) illustrated that the OK cells used inthis study were capable of receptor-mediated endocytosis. Thespecificity of the latter was demonstrated by competition for theuptake of 50–100 µg/ml albumin–FITC upon simultaneousincubation with a 25-fold excess of bovine serum albumin (BSA) orincubation performed at 4�C. A similar internalization of dextran–FITC–lysine demonstrated fluid-phase endocytosis capacity of theOK cells used (data not shown). Endogenous expression ofCLC-5 in the OK cells was detected by immunofluorescence.Thus, a diffuse, finely punctate cytoplasmic pattern compatiblewith endosomal reactivity was observed in OK cells fixed incontrol conditions (Fig. 6D and inset); the pattern was moreconcentrated near the plasma membrane when cells were fixedduring endocytosis (Fig. 6E and inset). The much fainter,perinuclear signal observed with the pre-immune serum (Fig. 6F)is due to non-specific binding of the secondary FITC-labelledantibody, as it was also observed in the absence of the primaryantibody. These results obtained with the L8-9 antiserum (Fig. 6)were similar to those obtained with the L1-2 antiserum and theaffinity-purified antibodies (data not shown).

Co-localization studies were performed after 5 min incubationwith the two endocytosis tracers (Fig. 6G and H). When the signalwas combined with immunofluorescence for CLC-5, it was clearthat CLC-5 was not co-localized with the endocytic vesiclescontaining dextran–FITC (Fig. 6G). In contrast, a substantialfraction of the CLC-5 intracellular pattern was co-localized withthe endocytic vesicles containing albumin–FITC (Fig. 6H). Theco-localization between CLC-5 expressed near the cell membrane(Fig. 6I) and albumin–FITC (Fig. 6J) was confirmed at higher

magnification (Fig. 6K). These results suggest that the localizationof CLC-5 in the proximal renal tubular model of the OK cells iswith the endosomes involved in the receptor-mediated pathwaythat transports albumin.

DISCUSSION

Our results, which represent the first intra-renal localization ofCLC-5 in the human kidney, demonstrate that CLC-5 is expressedat multiple sites in the nephron. Thus, CLC-5 is expressedconsistently in the epithelial cells of the proximal renal tubule andthe thick ascending limb of Henle’s loop, and in intercalated cellsof the collecting duct. In addition, our results reveal that CLC-5expression is intracellular and that its subcellular distribution iscompatible with recycling early endosomes. Our further studiesusing the proximal tubular cell model of OK cells (20) suggest thatthis expression of CLC-5 occurs specifically in the endosomes thatform part of the receptor-mediated endocytic pathway, whichtransports albumin and low molecular weight proteins. Theseresults reveal a pathophysiological explanation for the renaltubular abnormalities observed in Dent’s disease, which is due tomutations that result in a functional loss of CLC-5.

These observations are based upon the detection of CLC-5expression by our specific antisera, and affinity-purified anti-bodies, raised against two epitopes (Fig. 1). However, CLC-5 hasa significant homology to CLC-3 and CLC-4 and, in order todemonstrate the high specificity of our antisera raised againstCLC-5, we undertook several investigations as follows. Thus, inaddition to establishing the specific recognition of the in vitrotranslated CLC-5 proteins (Fig. 1B) by both antisera, we obtainedthree lines of evidence to demonstrate the lack of cross-reactivityby: (i) the dot-blot analyses using peptides from CLC-3, CLC-4and CLC-5 (Fig. 1C); (ii) the tissue expression pattern for CLC-5(Fig. 1D) which demonstrated a predominant reactivity in the

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Figure 4. Immunohistochemical localization of CLC-5 and reference antigens in the human kidney cortex (A–D and N) and medulla (E–M and O). (A) Staining forCLC-5 (CLC-5 antiserum L1-2) was located in the proximal tubules in the human kidney cortex. The glomeruli were unstained, as were the connecting and distaltubules (×110). (B) Within proximal tubular epithelial cells, the staining pattern for CLC-5 was diffusely intracellular, mostly located above nuclei (×225). This patternwas different from that of vacuolar H+-ATPase (1:50 dilution), shown in (C), which was more polarized and apical (×165). (D) Adsorption of CLC-5 antiserum L1-2with the cognate peptide abolished staining of the human kidney cortex (×80). (E) In human kidney medulla, CLC-5 was located in the thick ascending limbs of Henle’sloop, while the majority of collecting duct profiles were unstained (×225). (F) The staining pattern for CLC-5 in the epithelial cells lining the thick ascending limbswas similar to that observed in the proximal tubules (B) and was mostly intracellular. A faint, inconsistent staining for CLC-5 was also observed in the thin descendinglimbs of Henle’s loop (×275). Identification of the latter segment was confirmed by immunolocalization for the water channel AQP1 (G) (×175). (H and I) Serialsections of human kidney medulla stained for CLC-5 (L1-2 antiserum) (H) or for the Tamm–Horsfall protein (I), which is a specific marker of the thick ascendinglimbs (18). The co-localization of CLC-5 and the Tamm–Horsfall protein further demonstrated CLC-5 expression in thick ascending limbs of Henle’s loop (×200).(J) Specific staining in the human kidney medulla was not detected when incubation was performed with antiserum L1-2 pre-adsorbed with the cognate peptide (×175).(K) Intercalated cells also stained for CLC-5, as shown on transverse sections of the medullary collecting ducts (×275). (L and M) Serial sections demonstratingco-localization of CLC-5 (L) in intercalated cells stained for H+-ATPase (M). CLC-5 immunoreactivity was found mostly in the α-type (asterisk) intercalated cells,which are acid secreting and contain apical H+-ATPase (×250). (N and O) An absence of specific staining in human kidney cortex (N) and medulla (O) was observedwith the pre-immune serum at the same dilution (×225).

human kidney, as compared with the lack of expression in the brainand skeletal muscle, which express most of CLC-3 (21) and CLC-4(22), respectively; and (iii) the studies using antisera pre-absorbedwith the cognate CLC-5 peptide or with the corresponding CLC-3and CLC-4 peptides (Figs 2B, 4D and J and 5C).

Our finding of CLC-5 expression in the human proximal tubuleis in agreement with that observed in rat kidney by RT–PCR studiesof micro-dissected tubules (15) and immunohistochemistry (16). Inaddition, our observation of the intracellular pattern of CLC-5expression, which is concentrated in the cytoplasm above the

nuclei but beneath the brush border (Fig. 4), is similar to thatreported in the rat (16). Our subcellular localization studies usinghuman kidney fractions (Fig. 2) further support these findings andindicate a role for CLC-5 in endosomes. Thus, repeatedcharacterization of subcellular fractions obtained from the cortexof different human kidneys revealed that the CLC-5 distributionparalleled that of Rab4, which is a marker of the early endosomeand plasma membrane recycling pathway. This cellular andsubcellular localization of CLC-5 in the human proximal tubule,which is the major site for the endocytic reabsorption of albumin

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Figure 5. Expression of CLC-5 in mammalian kidneys and in cultured renal cells. (A) RT–PCR analysis of CLC-5 mRNA products revealed the expected 315 bpproduct from exons 4–6. CLC-5 mRNA was detected by the use of human CLC-5 primers in mouse (Mo), rat (Ra) and human (Hu) kidneys, and in OK and HEK293cells. N1–N3 indicate Epstein–Barr virus-transformed lymphoblastoids from three normal individuals. The RT–PCR product was not present when only genomic DNA(G) or a water blank (B) were used, thereby demonstrating that this product is specific for RNA and is not due to amplification of a genomic sequence. The standardsize marker (S) in the form of a 1 kb ladder is shown. (B) Western blot analysis of CLC-5 in cell lysates from OK cells. Thirty micrograms of total protein were loaded(7.5% gel) and the blots were probed with the anti-CLC-5 L8-9. Pre-immune (lane 1) and immune sera (lane 2) at the same dilution were used. An 83 kDa band,corresponding to the predicted molecular mass of CLC-5, was identified (arrow). Additional bands of much lower molecular weight were also detected with thepre-immune serum. The 83 kDa band was also identified with the L1-2 antiserum and with both of the affinity-purified antibodies (data not shown). The films wereexposed for 30 s. (C) The specificity of the 83 kDa band detected with the CLC-5 antiserum L8-9 was tested by peptide adsorption. OK cell lysate (30 µg of total proteinper lane) was electrophoresed (7.5% gel) and transferred to nitrocellulose. Following demonstration of similar transfer efficacy by Ponceau red staining, nitrocellulosestrips were cut and probed with: (i) CLC-5 antiserum L8-9 (lane 1); (ii) L8-9 antiserum pre-adsorbed with the cognate CLC-5 peptide (lane 2) or the corresponding,CLC-4 peptide (lane 3); and (iii) the pre-immune serum L8-9 (lane 4). The intensity of the 83 kDa band was markedly reduced by specific peptide adsorption (lane 2)but not when adsorption was performed with the control peptide (lane 3). No signal was observed with pre-immune serum (lane 4). The films were exposed for 30 s.The molecular mass standards are given in kDa.

and low molecular weight proteins, together with the phenotypeof Dent’s disease, indicates a role for CLC-5 in the endosomalpathway mediating proximal tubular protein reabsorption. Thishypothesis is supported partially by in vitro studies ofCLC-5-transfected distal tubular canine (MDCK) and Africangreen monkey (COS-7) cells which have revealed CLC-5co-localization with endocytosed proteins in endosomes (16).However, a cautious extrapolation of these findings is required asMDCK and COS-7 cells are not proximal tubular cells and theirrole in the tubular reabsorption of proteins, e.g. albumin, has notbeen established (34). In order to elucidate the putative role ofCLC-5 in the proximal tubular reabsorption of proteins by anendocytic pathway, we pursued in vitro studies using OK cells,which are an established model for proximal tubular cells (20,32).Our results demonstrate the endogenous expression of CLC-5 inOK cells, and co-localize CLC-5 to endosomes that are involvedin the receptor-mediated endocytosis of albumin (Figs 5 and 6).These data suggest a possible explanation for the low molecularweight proteinuria observed in Dent’s disease, as follows. Adecreased flow of chloride ions, due to CLC-5 dysfunction, intothe endocytic vesicles, which are acidified by the H+-ATPase (31),would diminish the supply of counter-ions for the transportedprotons. This in turn would limit acidification and result in

inefficient reabsorption of low molecular weight proteins andalbumin (32). It is of interest to note that, in this receptor-mediatedendocytic pathway, the receptor that binds albumin and lowmolecular weight proteins is megalin (35,36), a glycoprotein, alsoreferred to as gp-330 and related to the low density lipoprotein(LDL) receptor (37). Megalin also binds other ligands, notablycalcium ions, and it may be responsible for the large portion ofnon-regulated calcium uptake that occurs in the proximal tubule(32). Hence the putative co-localization of megalin and CLC-5,as inferred from the localization of CLC-5 in albumin-containingendosomes (Fig. 6), suggests the intriguing possibility that thistype of endosome may mediate transcellular calcium reabsorption inthe proximal tubule. CLC-5 mutations may then possibly impairendosomal trafficking (13) and thereby provide a unifyingexplanation for two of the major features of Dent’s disease (1,2),notably hypercalciuria and low molecular weight proteinuria.

The localization of CLC-5 to the thick ascending limb ofHenle’s loop of human kidney (Fig. 4E, F, H and I), which wasnot observed in similar studies of the rat (16), is a novel findingthat may be of importance in the aetiology of the hypercalciuriaof Dent’s disease. This may be of further interest as a role in cationhomeostasis has also been demonstrated for the yeast CLC (38).However, our finding of CLC-5 expression in the human thick

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Figure 6. Albumin endocytosis and endogenous expression of CLC-5 in OK cells: confocal microscopy. (A–C) Albumin endocytosis was studied after incubationof OK cell monolayers with 10 mg/ml albumin–FITC and imaging by confocal microscopy after 2 (A), 5 (B) and 15 min (C). Fluorescent endocytosis vesicles appearednear the margins of the cells after the 2 min incubation and extended deeper into the cytoplasm after 5 and 15 min incubation (×200). (D–F) Endogenous CLC-5 wasdetected by immunofluorescence (antiserum L8-9, 1:100) on subconfluent monolayers of OK cells fixed in control conditions (D and inset) or during endocytosis(15 min incubation with BSA) (E and inset). A diffuse, punctate intracellular staining was observed in both conditions. Note that the immunoreactivity for CLC-5 wasmore concentrated near the cell plasma membrane during endocytosis (compare insets; n, nucleus). Only a faint, paranuclear staining was detected when parallel OKcell monolayers were incubated with either the pre-immune serum at the same dilution (F) or without the primary antibody (data not shown). The confocal settingswere identical for (D–F) (D and E ×250, insets ×500, F ×200). (G–K) Examination of the co-localization between CLC-5 (red) and tracers (green), dextran–FITC oralbumin–FITC, used preferentially to follow fluid-phase or receptor-mediated endocytosis vesicles, respectively. Following a 5 min incubation of the monolayers at37oC with either dextran–FITC–lysine (G) or albumin–FITC (H), endogenous CLC-5 in OK cells was detected with the L8-9 antiserum followed by a TRITC-coupledsecondary antibody. No co-localization between CLC-5 and endocytic vesicles containing dextran–FITC was detected after 5 min uptake (G). In contrast, after a similaruptake time, the emission of the two fluorochromes (yellow) in the same cytoplasmic structures (H) suggested that a substantial fraction of CLC-5 was co-localizedwith the albumin–FITC vesicles (×325). At higher magnification, the co-localization between CLC-5 expressed near the cell membrane (I) and an albumin–FITCvesicle (J) is illustrated by the yellow emission (K) (×750). The image was obtained after 10 min endocytosis of 100 µg/ml albumin–FITC.

ascending limbs of Henle’s loop may require cautious interpretation,as CLC-Ka and CLC-Kb, which share <10% overall identity overthe epitope regions, are also known to be present in the basolateralmembrane of the epithelial cells lining the thick ascending limbs(39). Cross-immunoreactivity between these and CLC-5 is apossible explanation, but the <10% identity between the CLC-5and CLC-Ka and Kb epitopes (compared with the >65% identitybetween CLC-5 and CLC-3 and CLC-4) together with the

competition experiments, performed both for immunostainingand immunoblotting, indicate a high specificity for the CLC-5antisera and make this possibility unlikely. In addition, the diffusestaining pattern observed for CLC-5 is markedly different fromthe membrane pattern reported for CLC-Ka (39). Thus, it seemslikely that CLC-5 is expressed in the epithelial cells of the thickascending limb of Henle’s loop, which also express the calcium-sensing receptor (40), and the situation may be analogous to that

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reported for the aetiology of the hypercalciuria in the differentforms of Bartter’s syndrome (9), in which mutations involvingthe bumetanide sensitive Na+-K+-Cl– co-transporter (NKCC2),the inwardly rectifying renal potassium channel (ROMK) orCLC-Kb interact, by mechanisms that need to be defined fully,with the calcium-sensing receptor (CaR) to regulate calciumreabsorption (7,9,10,41).

CLC-5 was also found to be expressed in intercalated cells ofthe collecting duct (Fig. 4K), and this finding is consistent withthat reported by in situ hybridization and immunohistochemicalstudies of rat kidney sections (14,16). Our co-localization studiesperformed with H+-ATPase revealed that intercalated cells, themajority of which were of the α type, express CLC-5 at adetectable level in the human kidney (Fig. 4L and M). The roleof CLC-5 in these intercalated cells may be associated with thechloride–bicarbonate exchangers (42) and/or a conductive chloridepathway in these cells (43). Thus, it has been proposed that lossof CLC-5 function due to Dent’s disease mutations may lead toa lack of chloride ion flow and an accompanying dysfunction ofproton excretion by the α-type intercalated cells (14). This in turnmay lead to the impaired urinary acidification defect observed inDent’s disease, and the more alkaline environment coupled to thehigher urinary phosphate and protein losses may result inenhanced renal stone formation in patients with Dent’s disease(1,2).

In conclusion, we have raised and characterized antisera againsthuman CLC-5. Western blot and immunostaining experimentsperformed in human kidneys have shown that CLC-5 is locatedat multiple sites in the nephron which include the proximal renaltubules, the thick ascending limbs of Henle’s loop and intercalatedcells of the collecting duct. The subcellular expression of CLC-5is compatible with its location in the endosomes that form a partof the receptor-mediated endocytic pathway which transportsalbumin. These results will help to elucidate further the putativefunction(s) of CLC-5 and its role in the pathophysiology of Dent’sdisease.

MATERIALS AND METHODS

Tissue samples, cell cultures and fractions

A total of 11 samples from normal adult kidneys (mean age42 years, range 16–77 years) were obtained directly at surgery.Nine samples were cadaveric kidneys prepared for transplantation(but not used for technical reasons), and two originated from theopposite, tumour-free pole of kidneys removed for polar hyper-nephroma. All samples were perfused with ice-cold neutralbuffered salt solutions and kept at 4�C prior to use (44). Autopsysamples, within 5 h post-mortem, were also obtained frompancreas, liver, brain, kidney and skeletal muscle. Placenta wasfreshly obtained. The use of these human samples had beenapproved by the University of Louvain Ethical Review Board.OK cells (passage 88–93) and human embryonic kidney(HEK)293 cells were cultured using appropriate conditions(45,46). Membrane and subcellular fractions were prepared fromfour adult human kidneys and other tissues, as previouslydescribed (44,46). Briefly, kidney samples were homogenizedwith a Potter apparatus in ice-cold buffer (300 mM sucrose and25 mM HEPES made to pH 7.0 with 1 M Tris), containingprotease inhibitors. The homogenate was centrifuged at 1000 gfor 20 min at 4�C, and the resulting supernatant was centrifugedat 80 000 g for 30 min at 4�C. The pellet, representing the

membrane fraction, was suspended in ice-cold homogenizationbuffer, or solubilized with 0.5% CHAPS (Pierce, Rockford, IL)(46). Subcellular fractionation based on differential centrifugationwas performed (24,25) on three human kidneys (Fig. 3). Briefly,the kidney homogenate was centrifuged at 19 000 g for 20 min at4�C. The initial pellet was layered on top of a 1.12 M sucrosesolution, and centrifuged at 100 000 g for 60 min at 4�C. Theresulting pellet contained nuclei and mitochondria. The layer atthe interphase was centrifuged again at 40 000 g for 20 min at 4�Cto give a final pellet [plasma membrane (PM) fraction]. Twoadditional subcellular fractions were collected by differentialcentrifugation of the initial supernatant: a low-speed pellet (LSP)after 41 000 g for 20 min at 4�C, with corresponding supernatant(LSS), that was separated further into a high-speed pellet (HSP)after 160 000 g for 75 min at 4�C, with a correspondingsupernatant (HSS). Protein concentrations were determined withthe bicinchoninic acid (BCA) protein assay (Pierce), using BSAas standard (44). The extraction yield in the five fractions (LSP,LSS, HSP, HSS and PM) averaged 2.6, 0.9, 27.8, 1.4 and21.4 µg/mg tissue, respectively. All extracts were snap frozen inliquid nitrogen and stored at –80�C until further use.

Antisera

Rabbit antisera directed against human CLC-5 were raised againstsynthetic peptides, VTFEERDKCPEWNSWSQL (correspondingto residues 108–125; Fig. 1, L1-2), and SELISELFNDCGLLDSSK(corresponding to residues 382–399; Fig. 1, L8-9). The peptideswere conjugated to BSA using glutaraldehyde, and four rabbitswere injected with each immunogen which consisted of anemulsion of the conjugate solution and Freund’s adjuvant.Corresponding peptides from CLC-3 (residues 121–138 and395–412) and CLC-4 (residues 121–138 and 395–412), whichwere 67 and 72%, and 67 and 83% identical, respectively, werealso synthesized to assess the specificity of the CLC-5 antibodies.Specificity of both anti-CLC-5 sera was assessed by adsorptionwith the cognate peptide or the corresponding peptides fromCLC-3 or CLC-4 (1 µg peptide/µg antiserum protein). Bothantisera were affinity purified using the relevant peptide attachedto a cyanogen bromide-activated Sepharose column (AmershamPharmacia Biotech, Uppsala, Sweden). In addition, rabbitpolyclonal antibodies against Rab4, Rab5a and Rab6 isoforms(Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonalantibody raised against β-COP (Sigma, St Louis, MO), amonoclonal antibody, E11, raised against the 31 kDa subunit ofthe vacuolar H+-ATPase (30), an affinity-purified antibodyagainst human aquaporin-1, AQP1 (47) and a sheep polyclonalantibody against Tamm–Horsfall protein (Biodesign International,Kennebunk, ME) were used.

Western blot analysis

Protein extracts from in vitro translation products, tissue extractsand kidney fractions and OK cells were separated by SDS–polyacrylamide gels and transferred to nitrocellulose or PVDF aspreviously described (46). In vitro translation of CLC-5 wasperformed using cDNAs cloned in PTLN (2) and the TnT-coupled wheat germ extract system (Promega, Madison, WI).After blocking, membranes were incubated overnight at 4�C withprimary antibodies (1:1000 dilution), washed, incubated for 1 hat room temperature with the appropriate anti-rabbit IgG oranti-mouse IgG peroxidase-labelled antibody (1:5000; Dako,

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Glostrup, Denmark), washed again, and visualized after 1 minincubation with enhanced chemiluminescence (ECL; AmershamPharmacia Biotech). Ponceau red (Sigma) staining was performedsystematically to assess transfer efficiency. The experiments wereperformed and verified on a minimum of two occasions.Specificity of the immunoblot was determined by incubation with(i) pre-immune rabbit serum; (ii) non-immune rabbit or controlmouse IgG (Vector Laboratories, Burlingame, CA) or controlascites fluid (Sigma); and (iii) pre-adsorbed anti-CLC-5 antisera.

RNA extraction, RT–PCR and gel electrophoresis

RNA was extracted from tissues, OK cells and HEK293 cells, andused with CLCN5 nested primers (outer primer pair: 5′-TGCTG-TTCTGGTTTAAACCATGAAC-3′ and 5′-CTCAAGCCAGA-CGACAGTGCC-3′, and inner primer pair 5′-ACCATGAACAT-TGTTGCTGGAAC-3′ and 5′-GTGCCAGCACCAAGGTGAT-GG-3′) in RT–PCR as described (2) to detect CLCN5 expression.

Immunostaining

Tissue blocks were prepared from three normal, freshly obtainedhuman kidneys as described (44,46). Briefly, kidney sampleswere fixed for 6 h at 4�C in 4% paraformaldehyde (BoehringerIngelheim, Heidelberg, Germany) in 0.1 M phosphate buffer,pH 7.4, prior to embedding in paraffin. Sections 6 µm thick wererehydrated and incubated for 30 min with 0.3% hydrogenperoxide to block endogenous peroxidase. Following incubationwith 10% normal goat serum in phosphate-buffered saline (PBS)for 20 min, sections were incubated for 45 min with the primaryantibodies (dilution 1:100) in PBS containing 2% BSA. Afterthree washes of 5 min each, sections were incubated with theappropriate biotinylated secondary anti-IgG goat antibodies(Vector Laboratories), washed again and incubated for 45 minwith the avidin–biotin peroxidase complex (Vectastain Elite;Vector Laboratories). After three more washes of 5 min each,antibodies were detected using diaminobenzidine or aminoethyl-carbazole (Vector Laboratories). Immunolocalization was alsoperformed on acetone-fixed frozen sections prepared from twonormal adult human kidneys. All incubations were at roomtemperature in a humidified chamber. Control experimentsincluded incubation: (i) in the absence of primary antibody;(ii) with the pre-immune serum; (iii) with control rabbit or mouseIgG (Vector Laboratories); (iv) with unrelated antibodies; and(v) with pre-adsorbed anti-CLC-5 antisera. Sections weremounted and viewed under a Zeiss Axiophot 2 photomicroscope.

CLC-5 expression, endocytosis and co-localization studiesin OK cells by confocal microscopy

Endogenous CLC-5 expression was detected by immuno-fluorescence in OK cells seeded at a density of 2 × 104 cells/mlover glass coverslips. The cells were fixed for 10 min in 4%paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 20�C,under control conditions or following 15 min incubation at 37�Cwith 100 µg/ml BSA. After two 5 min washes in PBS-Ca2+, cellswere permeabilized with 0.5% Triton X-100 in PBS-Ca2+ for15 min. Quenching was performed by incubation with 0.1%glycine in PBS-Ca2+ for 20 min at room temperature, prior toincubation with the primary antibody diluted in PBS-Ca2+

containing 0.1% BSA for 1 h at room temperature. After being

washed four times for 5 min each in PBS-Ca2+ containing 0.1%BSA, the cells were incubated with the appropriate FITC- ortetramethylrhodamine isothiocyanate (TRITC)-labelled antibody(1:200; Sigma) for 1 h at room temperature. The coverslips werewashed again four times for 5 min each in PBS-Ca2+ containing0.1% BSA, and mounted with Mowiol/DABCO (Sigma) forviewing under a Bio-Rad MRC 1024 laser scanning confocalimaging system coupled to a Zeiss Axiovert 135M invertedmicroscope. The specificity of immunofluorescence was testedby incubation as follows: (i) in the absence of primary antibody;(ii) with pre-immune rabbit serum; (iii) with non-immune rabbitor mouse sera (Vector Laboratories); and (iv) with pre-adsorbedanti-CLC-5 antisera. Functional studies of endocytosis andco-localization studies with CLC-5 were performed on subconfluentmonolayers of OK cells grown on glass coverslips at 37�C. Afterbeing washed twice for 2 min each in isotonic Ringer’s solution(130 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mMHEPES, adjusted to pH 7.4 with 1 M Tris) at 37�C, themonolayers were incubated at 37�C for 2, 5 and 15 min with 1 mlof either 70 kDa dextran–FITC–lysine (13 mg/ml; MolecularProbes, Eugene, OR) or albumin–FITC (50 µg to 20 mg/ml; Sigma)(20). The cells were rinsed with ice-cold Ringer’s solution and trans-ferred on ice, to be washed a further 10 times with ice-cold Ringer’ssolution. The monolayers were then fixed and permeabilized asmentioned above. The labelling for CLC-5 was then performedas described above, and the coverslips were mounted for confocalviewing using settings defined to avoid signal interferencesbetween the channels. All experiments were performed induplicate or more and compared with those obtained usingpre-immune sera as controls.

ACKNOWLEDGEMENTS

Normal human tissue samples were obtained from Professor J.-P.Squifflet and Dr J.-P. Cosyns (St Luc Academic Hospital,Brussels, Belgium), and from the National Disease ResearchInterchange (Philadelphia, PA). OK cells were kindly providedby Dr G. Friedlander (Université Xavier Bichat, Paris, France)and cultured by C. Lebeau. The antibodies against H+-ATPaseand AQP1 were gifts from Dr S. Gluck (Washington University,St Louis, MO) and Dr P. Agre (Johns Hopkins University,Baltimore, MD), respectively. The CLC-5 construct in PTLN wasa gift from T.J. Jentsch (ZMNH, Hamburg, Germany). Our thanksto P. Byfield, M. Ghattei, G.R. Williams and S.H.S. Pearce forhelp and advice in raising the antisera, and to S. Combet, M.Leruth, P. Moulin and P. Van der Smissen for help in someexperiments. We are grateful to the Fonds de la RechercheScientifique Medicale (convention 3.4566.97 and credit9.4531.94F), the Fonds National de la Recherche Scientifique(credit 9.4540.96) and the Medical Research Council (MRC) UK(P.T.C. and R.V.T.) for support.

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