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Human Molecular Genetics Pages 247-257  

Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent’s disease
Introduction
Results
   Characterization of CLC-5 antisera
   CLC-5 expression in the human kidney
   Immunolocalization of CLC-5 in the human kidney
   CLC-5 expression, and co-localization studies with endocytosis tracers in OK cells
Discussion
Materials And Methods
   Tissue samples, cell cultures and fractions
   Antisera
   Western blot analysis
   RNA extraction, RT-PCR and gel electrophoresis
   Immunostaining
   CLC-5 expression, endocytosis and co-localization studies in OK cells by confocal microscopy
Acknowledgements
References

Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent’s disease

Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent’s disease

Olivier Devuyst1, Paul T. Christie2, Pierre J. Courtoy3, Renaud Beauwens4 and Rajesh V. Thakker2,*

1Division of Nephrology and 3Cell Unit, Christian de Duve Institute of Cellular Pathology, University of Louvain Medical 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 of Physiopathology, 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 disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle’s loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent’s disease.

INTRODUCTION

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, result in Dent’s disease, which is a renal tubular disorder characterized by low molecular weight (<40 kDa) proteinuria (e.g. [alpha]1 and [beta]2 microglobins, lysozyme and retinol-binding protein), albuminuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and renal failure (1-4). In addition, other renal proximal tubular defects, which include aminoaciduria, phosphaturia, glycosuria, kaliuresis, uricosuria and an impairment of urinary acidification may also occur (1). CLCN5, which in man is expressed predominantly in the kidney, belongs to the family of mammalian voltage-gated chloride channel genes (CLCN1-CLCN7, CLCNKa and CLCNKb) that encode proteins (CLC-1-CLC-7, CLC-Ka and CLC-Kb) with ~12 transmembrane domains (Fig. 1) (5-7). These chloride channels are important for the control of membrane excitability, transepithelial transport and possibly regulation of cell volume. To date, only mutations of CLCN1, CLCNKb and CLCN5 have been reported to be associated with human disorders, and these are the myotonia disorders of Thomsen and Becker (8), a form of Bartter’s syndrome (9) and Dent’s disease (2,3), respectively (7). Heterologous expression of wild-type CLC-5 in Xenopus oocytes results in chloride conductance (2) which is markedly reduced or abolished by the CLC-5 mutations of Dent’s disease (2,3). However, the mechanisms whereby a loss of this chloride conductance leads to hypercalciuria and the proximal renal tubular defects, e.g. low molecular weight proteinuria, remain to be elucidated.

   A - C
   D

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 746 amino 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) channel is 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 two different 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% gels and 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 the wild-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 immunoreactivity to 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 and probed 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 detected by 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, which was 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).

Calcium reabsorption occurs in a large segment of the nephron that includes the proximal tubule, the thick ascending limb of Henle’s loop and the distal tubule (10), whilst that of albumin and low molecular weight proteins is restricted to the proximal tubule and involves endocytosis with subsequent transport to the acidic vacuolar-lysosomal system (11). A loss of CLC-5 function may decrease chloride influx in the endocytic vesicles and thereby prevent the dissipation of the charge that is generated by the electrogenic H+-ATPase pump for the provision of the acidic environment (6,11). This would lead potentially to impaired endosomal acidification and inefficient reabsorption of the low molecular weight proteins (11,12). In addition, a decreased reabsorption of chloride ions in the proximal tubule may also result in reduced calcium reabsorption (13). The identification of the specific segments of the nephron that express CLCN5 would help to clarify these and other mechanisms. Such studies have been performed in the rat kidney (14-16). Thus, in situ hybridization revealed CLCN5 expression consistently in the type [alpha] intercalated cells of the rat collecting duct but only occasionally in the proximal tubules (14). However, the use of RT-PCR on rat micro-dissected tubules revealed CLCN5 expression in all of the segments of the nephron, although expression in the glomerulus and the S2 segment of the proximal tubule was low (15). More recently, immunohistochemistry studies of rat kidney have localized CLC-5 expression to the proximal tubular cells and to both types of intercalated cells in the collecting duct (16). These different findings (14-16) in the rat nephron do not explain all the abnormalities observed in patients with Dent’s disease. In addition, any extrapolation of these findings from the rat to the human kidney requires caution, as there are significant developmental, functional and morphological differences between the two mammalian kidneys (17-19). Moreover, the tissue expression of CLCN5 in the two mammals also differs, with CLCN5 being expressed in the rat kidney, brain, lung and liver (15), but being expressed predominantly only in the human kidney (6).

In order to determine the segmental expression of CLC-5 in human kidney and to define its intracellular localization, we raised and characterized polyclonal antisera to two epitopes of CLC-5 (Fig. 1) and used them for expression studies using normal human kidney sections and subcellular fractions, respectively. In addition, we investigated further, by confocal microscopy, the intracellular distribution and putative function of CLC-5 by use of opossum kidney (OK) cells, which represent a well-established model for proximal tubular epithelial cells (20). Our results revealed that CLC-5 is localized to the endosomes and is expressed consistently in the human proximal tubule, the thick ascending limb of Henle’s loop and in the intercalated cells of the collecting duct. Thus, CLC-5 is expressed more widely in the human than the rat nephron, and our observed sites of CLC-5 expression help to provide further insight into the pathophysiological basis for the 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 putative loops (Fig. 1A), between D1 and D2, and D8 and D9, respectively, of human CLC-5, were characterized against in vitro translated wild-type CLC-5 and mutant W279X proteins (Fig. 1B). [35S]methionine incorporation (Fig. 1B) verified the efficiency of the in vitro translation, and western blot analysis using the L1-2 antiserum detected both the truncated and wild-type CLC5 proteins, whereas the L8-9 antiserum detected only the wild-type CLC-5 protein (Fig. 1B), as expected from the putative locations of their respective epitopes. In addition, an assessment of the specificity of L1-2 and L8-9 antisera, using the CLC-3, CLC-4 and CLC-5 peptides, together with the pre-immune and immune sera 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 be predominantly in the kidney and to a lesser extent in the placenta and pancreas; this is consistent with our previous observations of CLCN5 mRNA by northern blot analysis (6). This predominantly renal expression of CLC-5 is markedly different from that of CLC-3 and CLC-4 which are expressed at high levels in the brain (hippocampus and olfactory cortex) and skeletal muscle, respectively (21,22). Thus, the predominant renal expression of CLC-5 detected by the L1-2 and L8-9 CLC-5 antisera, and the lack of strong signals from extracts of the other human tissues, indicates that these antisera are specific for CLC-5 and do not have cross-reactivity to CLC-3 and CLC-4. Both antisera L1-2 and L8-9, which were used systematically in all of the studies, yielded similar results and identified the same structures by immunoblotting and immuno-staining. In addition, similar results were obtained using affinity-purified CLC-5 antibodies (Fig. 2A).


Figure 2. Western blot analysis and subcellular expression of CLC-5 in the human 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 kidneys were electrophoresed (7.5% gel) and transferred to nitrocellulose. The blots were probed with: (i) the anti-CLC-5 L8-9 (lanes 1-3); (ii) the corresponding pre-immune serum (lanes 4-6) at the same dilution (1:1000); or (iii) the affinity-purified L8-9 (lane 7) or L1-2 (lane 8) antibodies (1:100 dilution), and visualized 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 kDa band corresponding to the predicted molecular mass of human CLC-5 is identified specifically in all extracts (arrow). The second band at ~57 kDa is non-specific as it is also detected with the pre-immune serum. Similar results were also obtained with the L1-2 antiserum. (B) The specificity of the 83 kDa band detected with the anti-CLC-5 serum L8-9 in the human kidney was assessed by peptide adsorption. A human kidney extract (30 µg of total protein per 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-5 peptide (lane 2) or the corresponding peptide derived from CLC-3 (lane 3) or CLC-4 (lane 4); and (iii) the pre-immune serum L8-9 (lane 5). The intensity of the 83 kDa band (arrow) was markedly reduced by adsorption with the CLC-5 peptide (lane 2) but not affected with the CLC-3 or CLC-4 peptides (lanes 3 and 4). All films were exposed for 30 s. (C) Subcellular expression of CLC-5 in the human kidney. The five subcellular fractions of human kidney prepared by differential centrifugation (Fig. 3) were loaded (30 µg of total protein in each lane) and the blot was probed with the antiserum L8-9. The 83 kDa band corresponding to CLC-5 was detected with similar intensity in LSP and HSP fractions (lanes 1 and 3, arrowhead), and to a lesser extent in the PM fraction (lane 5). These fractions were investigated similarly using antibodies (dilution 1:200-1:500) raised against the Rab4 (14% gel), Rab5a (12% gel) and Rab6 isoforms (14% gel), [beta]-COP (7.5% gel) or vacuolar H+-ATPase (12% gel). The expression of Rab4, which was more abundant in the LSP and HSP than in the PM fractions, paralleled that of CLC-5, in contrast to that of [beta]-COP which was expressed almost exclusively in the HSP fraction. Rab5a and H+-ATPase were most enriched in the LSP fraction (lane 1). Representative immunoblots of at least two different experiments are shown.

CLC-5 expression in the human kidney

The L8-9 anti-CLC-5 serum identified two bands at 83 and ~57 kDa in human kidney extracts (Fig. 2A). The ~57 kDa band was detectable at similar densities with the pre-immune serum and was therefore considered to be non-specific for CLC-5. However, the 83 kDa band, which corresponded to the predicted molecular mass of the 746 amino acid CLC-5, was not detected by the pre-immune serum. In addition, this 83 kDa band was detected specifically by both (L1-2 and L8-9) of the affinity-purified antibodies (Fig. 2A) and by the L1-2 antiserum (Fig. 1D). Thus, the 83 kDa band was regarded as specific for CLC-5. A comparison of the signal intensities revealed that CLC-5 expression was higher in the renal cortex than in the medulla (Fig. 2A). The specificity for the 83 kDa band in human kidney extracts was confirmed further using peptide adsorption (Fig. 2B). The intensity of the 83 kDa band (lane 1) was markedly decreased when the L8-9 antiserum was pre-adsorbed with the cognate peptide of human CLC-5 (lane 2), but not affected when pre-adsorbed with a control peptide from human CLC-3 (lane 3) or CLC-4 (lane 4), while it was absent with the pre-immune serum L8-9 (lane 5). These results indicate that the CLC-5 antisera are specific for the renal expressed CLC-5 83 kDa band.

The subcellular distribution of CLC-5 expression was studied by using human kidney fractions (Fig. 2C) obtained by differential centrifugation (Fig. 3), as previously described for the isolation of subcellular fractions from liver and kidney (23-25). The distinct nature of these subcellular fractions (LSP, LSS, HSP, HSS and PM) was demonstrated by their differing protein compositions (assessed by SDS-PAGE and Coomassie staining), their differing vesicular content [assessed by electron microscopy (26)] and by their differing specific marker enzymes content (27). Fluid-phase endocytosis tracers such as dextran were located in both LSP and HSP fractions, whereas receptor-mediated endocytosis tracers such as albumin were primarily in the HSP fraction (24). Repeated immunoblot analyses showed that the 83 kDa band was equally abundant in the HSP and LSP, but consistently less abundant in the PM fraction (Fig. 2C). A faint band was also observed in the LSS (lane 2) upon longer exposure. A comparison of the distribution of CLC-5 immunoreactivity in these subcellular fractions with that of several markers of subcellular compartments was undertaken (Fig. 2C). These markers included: Rab4, which is localized throughout the early endosome and plasma membrane recycling pathway; Rab5a which is distributed in plasma membrane, clathrin-coated vesicles and early endosomes; Rab6, which is present in the Golgi stacks and trans-Golgi network (28); [beta]-COP, which is a marker of small vesicles of the secretory pathway (29); and H+-ATPase, the vacuolar subunit of the proton pump, which is detected in the plasma membrane and in clathrin-coated vesicles, and is concentrated in intracellular organelles including endosomes and lysosomes (30,31). Our analysis showed that the subcellular distribution of CLC-5 did not parallel that of [beta]-COP which is a marker for the Golgi complex that is enriched in the HSP fraction (Fig. 2C). In contrast, the distribution of CLC-5 was similar to that of H+-ATPase but was associated best with that of Rab4, thus being consistent with the early endosome and plasma membrane recycling pathway.


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

Immunolocalization of CLC-5 in the human kidney

Immunoreactivity for CLC-5 in the human nephron (Fig. 4) was detected in the cortical proximal tubules (Fig. 4A-D), the medullary thick ascending (Fig. 4E, F and H) and thin descending limbs (Fig. 4F) of Henle’s loop, and in intercalated cells of the collecting ducts (Fig. 4K and L). Comparable immunoperoxidase results, using paraformaldehyde kidney sections, were obtained with the L1-2 and L8-9 antisera, and with the affinity-purified antibodies. In addition, the use of specific antibodies facilitated the identification of: proximal tubules (vacuolar H+-ATPase); thin descending limbs (AQP1) and medullary thick ascending limbs (Tamm-Horsfall protein) of Henle’s loop; and intercalated cells of the collecting ducts (H+-ATPase). In the proximal renal tubules, CLC-5 expression was intracellular (Fig. 4B) and less polarized than that of H+-ATPase (Fig. 4C). This intracellular expression of CLC-5 was also observed in the medullary limbs of Henle’s loop (Fig. 4E, F and H). Controls that included pre-adsorbed antisera (Fig. 4D and J) and pre-immune sera (Fig. 4N and O) demonstrated the specificity of the immunoreactivity. Similar results were obtained by using immunofluorescence on acetone-fixed frozen sections from two normal human kidneys (data not shown).


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 for CLC-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 distal tubules (×110). (B) Within proximal tubular epithelial cells, the staining pattern for CLC-5 was diffusely intracellular, mostly located above nuclei (×225). This pattern was 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 L1-2 with 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’s loop, 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 limbs was 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 descending limbs of Henle’s loop (×275). Identification of the latter segment was confirmed by immunolocalization for the water channel AQP1 (G) (×175). (H and I) Serial sections 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 ascending limbs (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 demonstrating co-localization of CLC-5 (L) in intercalated cells stained for H+-ATPase (M). CLC-5 immunoreactivity was found mostly in the [alpha]-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 observed with the pre-immune serum at the same dilution (×225).

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

In order to investigate further the intracellular localization of CLC-5, studies were performed using OK cells which have been established as a model for proximal tubule epithelial cells (20,32). OK cells were demonstrated by RT-PCR to express the CLCN5 gene (Fig. 5A), and the presence of the 83 kDa CLC-5-specific band was demonstrated by immunoblot analysis (Fig. 5B and C). The putative endosomal localization of CLC-5 in OK cells was investigated further by confocal microscopy examination using dextran-fluorescein isothiocyanate (FITC)-lysine and albumin-FITC, which are two endocytic tracers that preferentially follow the fluid-phase and receptor-mediated endocytosis pathways, respectively (20,33). The time course of internalization of albumin-FITC (Fig. 6A-C) illustrated that the OK cells used in this study were capable of receptor-mediated endocytosis. The specificity of the latter was demonstrated by competition for the uptake of 50-100 µg/ml albumin-FITC upon simultaneous incubation with a 25-fold excess of bovine serum albumin (BSA) or incubation performed at 4°C. A similar internalization of dextran-FITC-lysine demonstrated fluid-phase endocytosis capacity of the OK cells used (data not shown). Endogenous expression of CLC-5 in the OK cells was detected by immunofluorescence. Thus, a diffuse, finely punctate cytoplasmic pattern compatible with endosomal reactivity was observed in OK cells fixed in control conditions (Fig. 6D and inset); the pattern was more concentrated near the plasma membrane when cells were fixed during 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-labelled antibody, as it was also observed in the absence of the primary antibody. These results obtained with the L8-9 antiserum (Fig. 6) were similar to those obtained with the L1-2 antiserum and the affinity-purified antibodies (data not shown).


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 bp product 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 HEK293 cells. 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 standard size 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 the pre-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 were exposed 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 protein per lane) was electrophoresed (7.5% gel) and transferred to nitrocellulose. Following demonstration of similar transfer efficacy by Ponceau red staining, nitrocellulose strips 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.


Figure 6. Albumin endocytosis and endogenous expression of CLC-5 in OK cells: confocal microscopy. (A-C) Albumin endocytosis was studied after incubation of 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 appeared near 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 was detected 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 was more concentrated near the cell plasma membrane during endocytosis (compare insets; n, nucleus). Only a faint, paranuclear staining was detected when parallel OK cell monolayers were incubated with either the pre-immune serum at the same dilution (F) or without the primary antibody (data not shown). The confocal settings were 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 or albumin-FITC, used preferentially to follow fluid-phase or receptor-mediated endocytosis vesicles, respectively. Following a 5 min incubation of the monolayers at 37oC 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-coupled secondary antibody. No co-localization between CLC-5 and endocytic vesicles containing dextran-FITC was detected after 5 min uptake (G). In contrast, after a similar uptake time, the emission of the two fluorochromes (yellow) in the same cytoplasmic structures (H) suggested that a substantial fraction of CLC-5 was co-localized with the albumin-FITC vesicles (×325). At higher magnification, the co-localization between CLC-5 expressed near the cell membrane (I) and an albumin-FITC vesicle (J) is illustrated by the yellow emission (K) (×750). The image was obtained after 10 min endocytosis of 100 µg/ml albumin-FITC.

Co-localization studies were performed after 5 min incubation with the two endocytosis tracers (Fig. 6G and H). When the signal was combined with immunofluorescence for CLC-5, it was clear that CLC-5 was not co-localized with the endocytic vesicles containing dextran-FITC (Fig. 6G). In contrast, a substantial fraction of the CLC-5 intracellular pattern was co-localized with the endocytic vesicles containing albumin-FITC (Fig. 6H). The co-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 localization of CLC-5 in the proximal renal tubular model of the OK cells is with the endosomes involved in the receptor-mediated pathway that transports albumin.

DISCUSSION

Our results, which represent the first intra-renal localization of CLC-5 in the human kidney, demonstrate that CLC-5 is expressed at multiple sites in the nephron. Thus, CLC-5 is expressed consistently in the epithelial cells of the proximal renal tubule and the thick ascending limb of Henle’s loop, and in intercalated cells of the collecting duct. In addition, our results reveal that CLC-5 expression is intracellular and that its subcellular distribution is compatible with recycling early endosomes. Our further studies using the proximal tubular cell model of OK cells (20) suggest that this expression of CLC-5 occurs specifically in the endosomes that form part of the receptor-mediated endocytic pathway, which transports albumin and low molecular weight proteins. These results reveal a pathophysiological explanation for the renal tubular abnormalities observed in Dent’s disease, which is due to mutations that result in a functional loss of CLC-5.

These observations are based upon the detection of CLC-5 expression by our specific antisera, and affinity-purified antibodies, raised against two epitopes (Fig. 1). However, CLC-5 has a significant homology to CLC-3 and CLC-4 and, in order to demonstrate the high specificity of our antisera raised against CLC-5, we undertook several investigations as follows. Thus, in addition to establishing the specific recognition of the in vitro translated CLC-5 proteins (Fig. 1B) by both antisera, we obtained three lines of evidence to demonstrate the lack of cross-reactivity by: (i) the dot-blot analyses using peptides from CLC-3, CLC-4 and CLC-5 (Fig. 1C); (ii) the tissue expression pattern for CLC-5 (Fig. 1D) which demonstrated a predominant reactivity in the human kidney, as compared with the lack of expression in the brain and skeletal muscle, which express most of CLC-3 (21) and CLC-4 (22), respectively; and (iii) the studies using antisera pre-absorbed with the cognate CLC-5 peptide or with the corresponding CLC-3 and CLC-4 peptides (Figs 2B, 4D and J and 5C).

Our finding of CLC-5 expression in the human proximal tubule is in agreement with that observed in rat kidney by RT-PCR studies of micro-dissected tubules (15) and immunohistochemistry (16). In addition, our observation of the intracellular pattern of CLC-5 expression, which is concentrated in the cytoplasm above the nuclei but beneath the brush border (Fig. 4), is similar to that reported in the rat (16). Our subcellular localization studies using human kidney fractions (Fig. 2) further support these findings and indicate a role for CLC-5 in endosomes. Thus, repeated characterization of subcellular fractions obtained from the cortex of different human kidneys revealed that the CLC-5 distribution paralleled that of Rab4, which is a marker of the early endosome and plasma membrane recycling pathway. This cellular and subcellular localization of CLC-5 in the human proximal tubule, which is the major site for the endocytic reabsorption of albumin and low molecular weight proteins, together with the phenotype of Dent’s disease, indicates a role for CLC-5 in the endosomal pathway mediating proximal tubular protein reabsorption. This hypothesis is supported partially by in vitro studies of CLC-5-transfected distal tubular canine (MDCK) and African green monkey (COS-7) cells which have revealed CLC-5 co-localization with endocytosed proteins in endosomes (16). However, a cautious extrapolation of these findings is required as MDCK and COS-7 cells are not proximal tubular cells and their role in the tubular reabsorption of proteins, e.g. albumin, has not been established (34). In order to elucidate the putative role of CLC-5 in the proximal tubular reabsorption of proteins by an endocytic 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 in OK cells, and co-localize CLC-5 to endosomes that are involved in the receptor-mediated endocytosis of albumin (Figs 5 and 6). These data suggest a possible explanation for the low molecular weight proteinuria observed in Dent’s disease, as follows. A decreased flow of chloride ions, due to CLC-5 dysfunction, into the endocytic vesicles, which are acidified by the H+-ATPase (31), would diminish the supply of counter-ions for the transported protons. This in turn would limit acidification and result in inefficient reabsorption of low molecular weight proteins and albumin (32). It is of interest to note that, in this receptor-mediated endocytic pathway, the receptor that binds albumin and low molecular weight proteins is megalin (35,36), a glycoprotein, also referred to as gp-330 and related to the low density lipoprotein (LDL) receptor (37). Megalin also binds other ligands, notably calcium ions, and it may be responsible for the large portion of non-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-containing endosomes (Fig. 6), suggests the intriguing possibility that this type of endosome may mediate transcellular calcium reabsorption in the proximal tubule. CLC-5 mutations may then possibly impair endosomal trafficking (13) and thereby provide a unifying explanation 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 of Henle’s loop of human kidney (Fig. 4E, F, H and I), which was not observed in similar studies of the rat (16), is a novel finding that may be of importance in the aetiology of the hypercalciuria of Dent’s disease. This may be of further interest as a role in cation homeostasis has also been demonstrated for the yeast CLC (38). However, our finding of CLC-5 expression in the human thick ascending limbs of Henle’s loop may require cautious interpretation, as CLC-Ka and CLC-Kb, which share <10% overall identity over the epitope regions, are also known to be present in the basolateral membrane of the epithelial cells lining the thick ascending limbs (39). Cross-immunoreactivity between these and CLC-5 is a possible explanation, but the <10% identity between the CLC-5 and CLC-Ka and Kb epitopes (compared with the >65% identity between CLC-5 and CLC-3 and CLC-4) together with the competition experiments, performed both for immunostaining and immunoblotting, indicate a high specificity for the CLC-5 antisera and make this possibility unlikely. In addition, the diffuse staining pattern observed for CLC-5 is markedly different from the membrane pattern reported for CLC-Ka (39). Thus, it seems likely that CLC-5 is expressed in the epithelial cells of the thick ascending limb of Henle’s loop, which also express the calcium-sensing receptor (40), and the situation may be analogous to that reported for the aetiology of the hypercalciuria in the different forms of Bartter’s syndrome (9), in which mutations involving the bumetanide sensitive Na+-K+-Cl- co-transporter (NKCC2), the inwardly rectifying renal potassium channel (ROMK) or CLC-Kb interact, by mechanisms that need to be defined fully, with the calcium-sensing receptor (CaR) to regulate calcium reabsorption (7,9,10,41).

CLC-5 was also found to be expressed in intercalated cells of the collecting duct (Fig. 4K), and this finding is consistent with that reported by in situ hybridization and immunohistochemical studies of rat kidney sections (14,16). Our co-localization studies performed with H+-ATPase revealed that intercalated cells, the majority of which were of the [alpha] type, express CLC-5 at a detectable level in the human kidney (Fig. 4L and M). The role of CLC-5 in these intercalated cells may be associated with the chloride-bicarbonate exchangers (42) and/or a conductive chloride pathway in these cells (43). Thus, it has been proposed that loss of CLC-5 function due to Dent’s disease mutations may lead to a lack of chloride ion flow and an accompanying dysfunction of proton excretion by the [alpha]-type intercalated cells (14). This in turn may lead to the impaired urinary acidification defect observed in Dent’s disease, and the more alkaline environment coupled to the higher urinary phosphate and protein losses may result in enhanced renal stone formation in patients with Dent’s disease (1,2).

In conclusion, we have raised and characterized antisera against human CLC-5. Western blot and immunostaining experiments performed in human kidneys have shown that CLC-5 is located at multiple sites in the nephron which include the proximal renal tubules, the thick ascending limbs of Henle’s loop and intercalated cells of the collecting duct. The subcellular expression of CLC-5 is compatible with its location in the endosomes that form a part of the receptor-mediated endocytic pathway which transports albumin. These results will help to elucidate further the putative function(s) of CLC-5 and its role in the pathophysiology of Dent’s disease.

MATERIALS AND METHODS

Tissue samples, cell cultures and fractions

A total of 11 samples from normal adult kidneys (mean age 42 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 the opposite, tumour-free pole of kidneys removed for polar hypernephroma. All samples were perfused with ice-cold neutral buffered salt solutions and kept at 4°C prior to use (44). Autopsy samples, within 5 h post-mortem, were also obtained from pancreas, liver, brain, kidney and skeletal muscle. Placenta was freshly obtained. The use of these human samples had been approved 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 from four adult human kidneys and other tissues, as previously described (44,46). Briefly, kidney samples were homogenized with a Potter apparatus in ice-cold buffer (300 mM sucrose and 25 mM HEPES made to pH 7.0 with 1 M Tris), containing protease inhibitors. The homogenate was centrifuged at 1000 g for 20 min at 4°C, and the resulting supernatant was centrifuged at 80 000 g for 30 min at 4°C. The pellet, representing the membrane fraction, was suspended in ice-cold homogenization buffer, or solubilized with 0.5% CHAPS (Pierce, Rockford, IL) (46). Subcellular fractionation based on differential centrifugation was performed (24,25) on three human kidneys (Fig. 3). Briefly, the kidney homogenate was centrifuged at 19 000 g for 20 min at 4°C. The initial pellet was layered on top of a 1.12 M sucrose solution, and centrifuged at 100 000 g for 60 min at 4°C. The resulting pellet contained nuclei and mitochondria. The layer at the interphase was centrifuged again at 40 000 g for 20 min at 4°C to give a final pellet [plasma membrane (PM) fraction]. Two additional subcellular fractions were collected by differential centrifugation 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 corresponding supernatant (HSS). Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay (Pierce), using BSA as standard (44). The extraction yield in the five fractions (LSP, LSS, HSP, HSS and PM) averaged 2.6, 0.9, 27.8, 1.4 and 21.4 µg/mg tissue, respectively. All extracts were snap frozen in liquid nitrogen and stored at -80°C until further use.

Antisera

Rabbit antisera directed against human CLC-5 were raised against synthetic peptides, VTFEERDKCPEWNSWSQL (corresponding to residues 108-125; Fig. 1, L1-2), and SELISELFNDCGLLDSSK (corresponding to residues 382-399; Fig. 1, L8-9). The peptides were conjugated to BSA using glutaraldehyde, and four rabbits were injected with each immunogen which consisted of an emulsion of the conjugate solution and Freund’s adjuvant. Corresponding peptides from CLC-3 (residues 121-138 and 395-412) and CLC-4 (residues 121-138 and 395-412), which were 67 and 72%, and 67 and 83% identical, respectively, were also synthesized to assess the specificity of the CLC-5 antibodies. Specificity of both anti-CLC-5 sera was assessed by adsorption with the cognate peptide or the corresponding peptides from CLC-3 or CLC-4 (1 µg peptide/µg antiserum protein). Both antisera were affinity purified using the relevant peptide attached to a cyanogen bromide-activated Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden). In addition, rabbit polyclonal antibodies against Rab4, Rab5a and Rab6 isoforms (Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal antibody raised against [beta]-COP (Sigma, St Louis, MO), a monoclonal antibody, E11, raised against the 31 kDa subunit of the vacuolar H+-ATPase (30), an affinity-purified antibody against human aquaporin-1, AQP1 (47) and a sheep polyclonal antibody against Tamm-Horsfall protein (Biodesign International, Kennebunk, ME) were used.

Western blot analysis

Protein extracts from in vitro translation products, tissue extracts and kidney fractions and OK cells were separated by SDS-polyacrylamide gels and transferred to nitrocellulose or PVDF as previously described (46). In vitro translation of CLC-5 was performed 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 with primary antibodies (1:1000 dilution), washed, incubated for 1 h at room temperature with the appropriate anti-rabbit IgG or anti-mouse IgG peroxidase-labelled antibody (1:5000; Dako, Glostrup, Denmark), washed again, and visualized after 1 min incubation with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Ponceau red (Sigma) staining was performed systematically to assess transfer efficiency. The experiments were performed 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 control mouse IgG (Vector Laboratories, Burlingame, CA) or control ascites 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, and used with CLCN5 nested primers (outer primer pair: 5[prime]-TGCTGTTCTGGTTTAAACCATGAAC-3[prime] and 5[prime]-CTCAAGCCAGACGACAGTGCC-3[prime], and inner primer pair 5[prime]-ACCATGAACATTGTTGCTGGAAC-3[prime] and 5[prime]-GTGCCAGCACCAAGGTGATGG-3[prime]) in RT-PCR as described (2) to detect CLCN5 expression.

Immunostaining

Tissue blocks were prepared from three normal, freshly obtained human kidneys as described (44,46). Briefly, kidney samples were fixed for 6 h at 4°C in 4% paraformaldehyde (Boehringer Ingelheim, Heidelberg, Germany) in 0.1 M phosphate buffer, pH 7.4, prior to embedding in paraffin. Sections 6 µm thick were rehydrated and incubated for 30 min with 0.3% hydrogen peroxide to block endogenous peroxidase. Following incubation with 10% normal goat serum in phosphate-buffered saline (PBS) for 20 min, sections were incubated for 45 min with the primary antibodies (dilution 1:100) in PBS containing 2% BSA. After three washes of 5 min each, sections were incubated with the appropriate biotinylated secondary anti-IgG goat antibodies (Vector Laboratories), washed again and incubated for 45 min with the avidin-biotin peroxidase complex (Vectastain Elite; Vector Laboratories). After three more washes of 5 min each, antibodies were detected using diaminobenzidine or aminoethylcarbazole (Vector Laboratories). Immunolocalization was also performed on acetone-fixed frozen sections prepared from two normal adult human kidneys. All incubations were at room temperature in a humidified chamber. Control experiments included incubation: (i) in the absence of primary antibody; (ii) with the pre-immune serum; (iii) with control rabbit or mouse IgG (Vector Laboratories); (iv) with unrelated antibodies; and (v) with pre-adsorbed anti-CLC-5 antisera. Sections were mounted and viewed under a Zeiss Axiophot 2 photomicroscope.

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

Endogenous CLC-5 expression was detected by immunofluorescence in OK cells seeded at a density of 2 × 104 cells/ml over 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°C with 100 µg/ml BSA. After two 5 min washes in PBS-Ca2+, cells were permeabilized with 0.5% Triton X-100 in PBS-Ca2+ for 15 min. Quenching was performed by incubation with 0.1% glycine in PBS-Ca2+ for 20 min at room temperature, prior to incubation 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- or tetramethylrhodamine isothiocyanate (TRITC)-labelled antibody (1:200; Sigma) for 1 h at room temperature. The coverslips were washed again four times for 5 min each in PBS-Ca2+ containing 0.1% BSA, and mounted with Mowiol/DABCO (Sigma) for viewing under a Bio-Rad MRC 1024 laser scanning confocal imaging system coupled to a Zeiss Axiovert 135M inverted microscope. The specificity of immunofluorescence was tested by incubation as follows: (i) in the absence of primary antibody; (ii) with pre-immune rabbit serum; (iii) with non-immune rabbit or mouse sera (Vector Laboratories); and (iv) with pre-adsorbed anti-CLC-5 antisera. Functional studies of endocytosis and co-localization studies with CLC-5 were performed on subconfluent monolayers of OK cells grown on glass coverslips at 37°C. After being washed twice for 2 min each in isotonic Ringer’s solution (130 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, adjusted to pH 7.4 with 1 M Tris) at 37°C, the monolayers were incubated at 37°C for 2, 5 and 15 min with 1 ml of either 70 kDa dextran-FITC-lysine (13 mg/ml; Molecular Probes, 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’s solution. The monolayers were then fixed and permeabilized as mentioned above. The labelling for CLC-5 was then performed as described above, and the coverslips were mounted for confocal viewing using settings defined to avoid signal interferences between the channels. All experiments were performed in duplicate or more and compared with those obtained using pre-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 Research Interchange (Philadelphia, PA). OK cells were kindly provided by Dr G. Friedlander (Université Xavier Bichat, Paris, France) and cultured by C. Lebeau. The antibodies against H+-ATPase and 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 was a gift from T.J. Jentsch (ZMNH, Hamburg, Germany). Our thanks to P. Byfield, M. Ghattei, G.R. Williams and S.H.S. Pearce for help and advice in raising the antisera, and to S. Combet, M. Leruth, P. Moulin and P. Van der Smissen for help in some experiments. We are grateful to the Fonds de la Recherche Scientifique Medicale (convention 3.4566.97 and credit 9.4531.94F), 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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*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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