Altered Polarity and Expression of H+-Atpase Without Ultrastructural

Kidney International, Vol. 63 (2003), pp. 1285–1295

CELL BIOLOGY – IMMUNOLOGY – PATHOLOGY

Altered polarity and expression of Hϩ-ATPase without ultrastructural changes in kidneys of Dent’s disease patients

PIERRE MOULIN,TAKASHI IGARASHI,PATRICK VAN DER SMISSEN,JEAN-PIERRE COSYNS, PIERRE VERROUST,RAJESH V. THAKKER,STEVEN J. SCHEINMAN,PIERRE J. COURTOY, and OLIVIER DEVUYST

Divisions of Pathology and Nephrology, and Cell Unit, Christian de Duve Institute of Cellular Pathology, Universite´ Catholique de Louvain, Brussels, Belgium; Department of Pediatrics, University of Tokyo, Tokyo, Japan; INSERM U538, CHU Saint Antoine, Paris, France; Molecular Endocrinology, Nufﬁeld Department of Medicine, University of Oxford, Oxford, United Kingdom; and Division of Nephrology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York

-Altered polarity and expression of H؉-ATPase without ultra- Mutations of the CLCN5 gene, located on chromo structural changes in kidneys of Dent’s disease patients. some Xp11.22, cause Dent’s disease, which is a renal tubu- Background. Dent’s disease is a proximal tubule (PT) disor- Ͻ der characterized by low-molecular-weight proteinuria (LWMP) lar disorder characterized by low-molecular-weight ( 60 that may be associated with hypercalciuria, nephrocalcinosis, kD) proteinuria (LMWP), albuminuria, hypercalciuria, and renal failure. It is caused by inactivating mutations of nephrocalcinosis, nephrolithiasis, and renal failure [1–3]. the renal chloride channel ClC-5, which colocalizes with the Other renal proximal tubular defects, including aminoacid- ϩ ␣ vacuolar H -ATPase in PT cells and -type intercalated cells. uria, phosphaturia, glycosuria, kaliuresis, and impairment Examinations of knockout mice have established the role of ClC-5 in PT endocytosis, but the consequences of ClC-5 muta- of urinary acidification, may also occur in some patients tions on the polarity of Hϩ-ATPase and other plasma mem- [1, 3]. CLCN5, which has a predominantly renal expres- brane proteins remain unknown. sion in humans and rodents, belongs to the CLCN family Methods. We have studied renal biopsies from eight patients of mammalian voltage-gated chloride channel genes that with Dent’s disease, due to inactivating ClC-5 mutations, by encode proteins (ClC-1 to ClC-7, ClC-Ka and ClC-Kb) light and electron microscopy, and by immunohistochemical staining. All patients exhibited LMWP, and renal function with 18 transmembrane domains [4]. These chloride chan- ranged from normal to end-stage renal failure. nels are important for the control of membrane excitabil- Results. Light microscopy revealed either normal renal ar- ity, transepithelial transport, and possibly regulation of chitecture or glomerulosclerosis, tubular dedifferentiation and cell volume [5]. Heterologous expression of wild-type atrophy, and mild interstitial fibrosis. Focal, hyaline casts, sometimes calcified, were identified at all stages. Electron mi- ClC-5 in Xenopus oocytes results in chloride conduc- croscopy did not reveal any ultrastructural abnormalities in tance, which is markedly reduced or abolished by the PT cells, and the endocytic apparatus was apparently normal. ClC-5 mutations of Dent’s disease [2, 5, 6]. However, immunohistochemical studies demonstrated a con- ϩ The segmental distribution of ClC-5 in the adult hu- sistent inversion of H -ATPase polarity in PT cells to a basolat- man kidney includes the proximal tubule (PT), the thick eral distribution contrasting with its apical location in the nor- ϩ ␣ mal kidney. This inversion of polarity was specific for H -ATPase ascending limb (TAL) of Henle’s loop and the -type and did not affect distribution of aminopeptidase, megalin, and intercalated cells (IC) of the collecting duct [7]. At the ϩ ϩ ϩ Na /K -ATPase. Furthermore, apical H -ATPase expression subcellular level, ClC-5 has been localized to PT apical ␣ was absent in -type intercalated cells. endosomes together with the vacuolar Hϩ-ATPase, there- Conclusion. ClC-5 mutations are associated with modifications in the polarity and expression of Hϩ-ATPase, but not by suggesting that it may contribute to the chloride ultrastructural alterations in PT cells. These findings help fur- conductance necessary for endosomal acidification and ther understanding of the role of ClC-5 and the pathophysiol- function [7–9]. Reabsorption of low-molecular-weight ogy of Dent’s disease. proteins by the PT depends on receptor-mediated endocytosis and transport to the acidic vacuolar-lysosomal Key words: chloride channel, ClC-5, intercalated cells, low-molecular- system [10]. Consequently, a loss of ClC-5 function may weight proteinuria, proton pump, proximal tubule. decrease chloride influx in the endocytic vesicles and thereby prevent the dissipation of the positive charge Received for publication February 19, 2002 ϩ and in revised form July 25, 2002, and September 25, 2002 generated by the electrogenic H -ATPase that is neces- Accepted for publication November 7, 2002 sary for the provision of the acidic environment [11].  2003 by the International Society of Nephrology This would lead to impaired endosomal acidification and

1285 1286 Moulin et al: The kidney in Dent’s disease inefﬁcient reabsorption (i.e., LMWP), which is a consis- NR tent feature of Dent’s disease [1, 3]. This hypothesis is NA supported by two independent studies of ClC-5–deﬁcient NA NR mice, which were shown to have a defect in receptor- NA NA

mediated endocytosis associated with LMWP and PT LM EM IS

dysfunction [12, 13]. These abnormalities in the ClC-5 c knockout mice were not associated with gross defects in Ϫ ϩϩϩ ϩϩ ϩϩϩ ϩϩ ϩϪ ϩϩϩ ϩϩϩ ϩϩϩ renal architecture, although the possibility remains that ϩ ϩϩϩ Nephro- there may be more speciﬁc ultrastructural abnormalities. calcinosis A, not available; NR, not reliable We have investigated renal histology and ultrastructure 250 mg/day). and immunolocalized several plasma membrane pro- Ͻ

ϩ ϩ teins, including the vacuolar H -ATPase, in the kidneys 2 of patients with Dent’s disease, all of whom had proven mg creatinine inactivating ClC-5 mutations. / 4 mg/kg/day ( Ͻ Urine dL mg mCa / 2 METHODS 8.0 880 mg/day NA 445 mg/day ␤ 20.7 4.4 mg/kg/day 14.2 NA Patients and controls, mutational analysis of CLCN5 mg b Ϫ ϩ ϩ ϩ / / /NA 9.8/ 6% / Renal biopsy samples were obtained from eight male /NA 7.6 37% ϩ ϩ Ϫ ϩ ϩ Ϫ

patients with Dent’s disease belonging to seven distinct G/AA pedigrees. Four of these patients originated from Univer- sity of Tokyo (Tokyo, Japan), three from State University dL / of New York (Syracuse, NY, USA), and one from Uni- 2.0 mg Plasma

versity College, London (UK). Seven of these eight pa- Creatinine tients have been previously reported [2, 14–17] to harbor 20%; normal urinary calcium excretion, a ClC-5 mutation (Table 1) and include two nonsense Ͻ (R648X, R28X), one missense (G506E), two acceptor splice-site mutations (intron 5, 132-172 deletion and ag to gg), and two deletions. Mutation analysis was performed in the fourth Japanese patient (# 4, Table 1), using CLCN5 speciﬁc primers for polymerase chain reaction (PCR) ampliﬁcation as previously described [15, 17]. This new mutation resulted from the deletion of a T ; # 5 [14]; # 6, # 7, and # 8 [2] have been described previously. Normal values: plasma creatinine, 0.1 to 0.8 mg/dL; adults, 0.6 (nucleotide 483) at codon 64 within exon 3 of CLCN5, leading to a frameshift and a stop codon (TAG) at codon 25% in mg/mg creatinine; adults, Ͻ , absence of protein 0.6 predicted effect ϩ

71 (Fig. 1). The four Japanese patients had a normal 2 → acid-base status at the time of renal biopsy (arterial blood → pH range, 7.38 to 7.41; plasma bicarbonate range, 23.5 Clinical and genetic characteristics of the eight male patients with Dent’s disease CLCN5 to 25 mEq/L). The age at renal biopsy in patients with frameshift and premature stop at codon 71 (D1) 0.3 NA 3.8 12% mutation →

Dent’s disease ranged from 6 to 54 years. The biopsy loss of D2 2.6

from patient # 8 was obtained at time of renal tranplanta- → tion for end-stage renal disease (ESRD). The principal Table 1. 0.025 mg/dL; urinary Ca abnormal splice variants and truncated protein 0.5 NA 1.6 28% clinical characteristics of the patients at the time of diag- Ͻ nostic evaluation are given in Table 1. Plasma creatinine → was normal in three Japanese patients (age Ͻ11 years) loss of 98 amino acids at C-terminus 1.1 disruption of charge distribution within D11 0.9 and slightly increased (1.1 mg/dL) in patient#1(14 premature stop at codon 28 (N-terminus) 2.2 → → years old). All patients tested had ϩ/ϩϩ proteinuria → ) of nephrocalcinosis was based on von Kossa’s staining

and LMWP. Hypercalciuria was present in ﬁve of seven Ϫ 180 kb deletion encompassing -microglobulin; D, transmembrane domain; EM, electron microscopy; G/AA, glycosuria/aminoaciduria; IS, immunostaining; LM, light microscopy; N 0.032 mg/dL; adults, 2 Ͼ ␤ patients assessed. Nephrocalcinosis was detected (von Ͻ m, m, a 2 2 ␤ Kossa’s staining) in one patient 6 years old and in all ␤ 9 T deletion at 483 (codon 64) 6 G506E 1410 R648X 11 Intron 5 ag to gg 3531 R28X G506E 54 Intron 5 132-172 deletion patients older than 10 years. An additional biopsy was years CLCN5 obtained at age 65 from the heterozygote carrier mother ) or absence ( ϩ e d d e d f of patient # 5; this female carrier had renal failure and e a long clinical history of hypertension. Samples from d Qualitative evaluation Patients originated from Japan The age at diagnosis was similar to age at renal biopsy, except for patientPatients # originated 8, from who the was United diagnosed States at age 42 Presence ( normal human kidney (renal transplant biopsies at time Patient originated from the United Kingdom Abbreviations are: a b c d e f #, code, origin # 5 (A-II-2) # 8 (19/94) # 6 (IV-13) # 2 (1/III-3) # 3 (9.3/95) # 7 (V-33) because of tubular damage/fixation.to Mutations 1.5 from mg/dL; patients urinary # 1 [15]; # 2 [16]; # 3 [17] Patient# 1 (1/2) Age of surgery, N ϭ 3; tumor-free pole of kidneys removed # 4 (1/II-1) Moulin et al: The kidney in Dent’s disease 1287

Fig. 1. Detection of the deletional mutation in exon 3 of CLCN5 in patient # 4. DNA sequence analysis of the affected male II-1 (A) revealed a T (nucleotide 483) deletion at codon 64, thus causing a frameshift that resulted in a missense peptide with a stop codon (TAG) at codon 71 (B and C). The mother I-1 (A) is heterozygous for the wild-type and mutant alleles (B). The mutations were confirmed by DNA sequence analysis of additional polymerase chain reaction (PCR) products. This DNA sequence abnormality was not detected in 110 alleles from 56 unrelated, normal Japanese individuals (54 females and 2 males), thereby indicating that this is not a common sequence polymorphism (data not shown). Individuals shown are male (box); female (circle); unaffected (open); carrier (half-filled); patient (filled).

for polar carcinoma, N ϭ 2) or immunoglobulin A (IgA) in paraffin. Six micrometer sections were cut and stained nephropathy (N ϭ 1) and minimal-change disease (N ϭ 1) with Masson’s trichrome. Calcium deposits in paraffin- were used for control experiments. The use of these hu- embedded sections were visualized with von Kossa’s stain- man samples has been approved by the University of ing [13]. For electron microscopy, samples were fixed in Louvain Ethical Review Board. 2.5% glutaraldehyde and postfixed in osmium tetroxide, before embedding in Epon. Ultrathin sections (80 nm), Processing for microscopy contrasted with uranyl acetate and lead citrate, were For light microscopy, samples were either frozen in examined through a Zeiss EM 109 (Carl Zeiss, Inc., New liquid nitrogen or fixed for 6 hours at 4ЊC in 4% formal- York, NY, USA) or a Philips CM12 (Philips, Eindhoven, dehyde in 0.1 mol/L phosphate buffer, then embedded The Netherlands) electron microscope. 1288 Moulin et al: The kidney in Dent’s disease

Subcellular fractionation Immunohistochemistry A sample from normal human kidney cortex was im- Immunostaining in human renal biopsies was per- mersed in ice-cold phosphate-buffered saline (PBS), and formed using immunoperoxidase, as described [7, 19]. carefully minced in 1 mm3 fragments that were homoge- Primary antibodies included the monoclonal antibody nized in 0.25 mol/L sucrose, 3 mmol/L imidazole, pH 7.0, against vacuolar Hϩ-ATPase [18]; rabbit polyclonal anti- by a Potter-Thomas homogenizer. Postnuclear particles bodies against aquaporin-1 (AQP-1) (Alamone, Jerusa- (denoted mitochondria-lysosomes-peroxysomes [MLP] lem, Israel); rabbit polyclonal antibodies against amino- fraction) were first retrieved by differential sedimenta- peptidase N [19]; sheep antibodies against megalin [21]; tion. For this purpose, nuclei and cell debris were re- a mouse monoclonal antibody against the Naϩ/Hϩ ex- moved by sedimentation at 10,000g ϫ minute, and this changer type 3 (NHE3) (Chemicon, Temecula, CA, USA); pellet was washed twice by sedimentation-resedimenta- and a mouse monoclonal antibody IVF12 against human tion under identical conditions. The pooled supernatants anion exchanger type 1 (AE1) [22]. Following incubation were further pelleted in a fixed-angle rotor (Ti 50, Beck- in 0.3% H2O2 (30 minutes) and 10% normal serum in man, Palo Alto, CA, USA) at 6 ϫ106g ϫ minute. After PBS (20 minutes) at room temperature, rehydrated sec- high-speed supernatant was removed, the high-speed tions were incubated successively with primary antibod- pellet was resuspended in 5 mL homogenization buffer ies; biotinylated goat antirabbit or horse antimouse IgG; by a Dounce homogenizer. This suspension was layered avidin-biotin peroxidase (ABP) complex; and, after wash- over a linear sucrose gradient (1.05 to 1.30 g/mL in den- ing, aminoethylcarbazole (Vector, Burlingame, CA, USA). sity) and equilibrated in a vertical rotor (vTi 50, Beck- Sections were examined under a Leica DMR coupled to man) at 24 ϫ 106g ϫ minute. Fourteen fractions of equal a Leica MPS60 photomicrographic system (Leica, Heer- volume were collected from the bottom (# 1, densest; brugg, Switzerland). The specificity of the immunolabel- # 13-14, loading zone) and analyzed for density (refrac- ing was confirmed by incubation without primary or sec- torimetry) and protein content using the BCA assay ondary antibody; or with nonimmune rabbit or mouse (Pierce Chemical, Rockford, IL, USA). Aliquots of IgG (Vector). equal volume were analyzed by Western blotting.

Western blotting RESULTS Sodium dodecyl sulfate-polyacrylamide gel electro- Light microscopy examination phoresis (SDS-PAGE) and immunoblotting were per- Light microscopy examination (Fig. 2) showed that formed as previously described [7]. Briefly, the extracts the Japanese patients (Fig.2AtoC),whowere all were solubilized by heating (95ЊC for 3 minutes) in sam- younger than 15 years old, had normal glomeruli and ple buffer [1.5% SDS, 10 mmol/L Tris-HCl, pH 6.8, 0.6% well-preserved tubules. The interstitium was free of in- dithiothreitol (DTT), and 6% (vol/vol) glycerol], before filtrate or fibrosis and arteries were normal. Hyaline casts separation on 7.5% acrylamide gels and transfer to nitro- were detected within the lumen of Henle’s loop (Fig. 2 B cellulose. The membranes were stained with Ponceau and C). The deposits were focal, without any interstitial Red (Sigma Chemical Co., St. Louis, MO, USA) to check change, and were not detected in the distal convoluted the efficiency of transfer, before blocking and incubation tubules or collecting ducts. They presented a red-to- with the primary antibody diluted in [2% bovine serum purple color gradient, compatible with early calcium de- albumin (BSA), 50 mmol/L phosphate buffer, 150 mmol/L position (Fig. 2C). Mild interstitial fibrosis was observed NaCl, 0.05% Tween 20, pH 7.4], at 4ЊC for 16 to 18 hours. in patient # 6 (31 years old and from the United States) The primary antibodies included a monoclonal antibody (Fig. 2D). The kidney of patient # 7 (6 years old and (E11) raised against the 31 kD subunit of the vacuolar from the United States) contained 14/58 glomeruli with Hϩ-ATPase [18]; an affinity-purified antibody raised complete or partial hyalinosis, the others showing slightly against the N-terminus of human ClC-5 [13]; rabbit poly- increased cellularity (Fig. 2E). Patients # 6 and # 7 dis- clonal antibodies against aminopeptidase N [19]; a mono- played mild tubular atrophy and hyaline casts located in clonal antibody against the ␣1-subunit of Naϩ/Kϩ-ATPase the outer medulla (Fig. 2 D to F). Some of the casts (Upstate Biotechnology, Lake Placid, NY, USA); and a were calcified, as indicated by von Kossa’s staining (Fig. monoclonal antibody against the lysosomal protein lyso- 2G). A mild interstitial fibrosis, predominantly located some-associated membrane protein 1 (LAMP1) [20]. The near arcuate arteries at the corticomedullary junction, membranes were washed, incubated for 1 hour at room was also detected. The kidney from the heterozygote temperature with the appropriate peroxidase-labeled an- female carrier had moderate or severe tubular atrophy tibody (Dako, Glostrup, Denmark), and visualized after and epithelial cell degeneration with many hyaline casts exposure to enhanced chemiluminescence (Amersham, in Henle’s loop, as well as a severe fibrosis throughout Arlington Heights, IL, USA). the biopsy (data not shown). Moulin et al: The kidney in Dent’s disease 1289

Fig. 2. Light microscopy findings in patients with Dent’s disease. (A) Representative section of the kidney cortex in patient # 2, with intact glomeruli, well-preserved tubular structures and arteries, and absence of fibrosis. (B and C) Outer medulla of patient # 4, in which hyaline casts are identified within the lumen of Henle’s loop (B). The gradient in dye affinity in the boxed field in (B) is shown at higher magnification in (C). (D) Inner cortex–outer medulla junction in patient # 6 showing mild interstitial fibrosis and foci of tubular basement membrane hyalinization and hyaline casts. (E to G) Representative sections from the inner cortex (E, G) and medulla (F) of patient # 7. (E) shows an almost hyalinized glomerulus, and a hyaline cast presenting a typical dye gradient within an atrophic tubule (arrow). (F) shows large areas of atrophic tubules with inflammatory infiltrate and hyaline cats. Calcifications within the interstitium are evidenced in black by von Kossa’s staining (G). (H to J) Sections from patient # 8. Medium-power view (H) of the end-stage kidney with interstitial nephritis features, adventitial fibrous thickening of blood vessels, and hyaline casts within atrophic tubules (arrows). von Kossa’s staining of a step section (I) demonstrates calcification of the hyaline globules. (J) illustrates at low-power large, confluent calcifications within atrophic areas. Masson’s trichrome, (A) to (F), and (H); von Kossa’s staining, (G), (I), and (J). Bars in (A), (B), (D) to (G), 50 ␮m; (C), 10 ␮m; (H) and (I), 100 ␮m; (J), 500 ␮m. 1290 Moulin et al: The kidney in Dent’s disease

Fig. 3. Conventional electron microscopy from patients with Dent’s disease. Patient # 3 has the entire CLCN5 gene deleted. Proximal tubule (PT) epithelial cells with normal morphology (A) that included a well-developed brush border, endosomal apparatus, numerous mitochondria, and thin basement membrane. Higher magniﬁcation illustrates a tall and well-differentiated brush border with preserved intercellular junctions (B), and normal abundance of apical endosomes (C). Intercellular junctions (D) are seen between PT epithelial cells in patient # 4. Cell boundaries are interdigitated, and well-differentiated tight junctions are evident. Bars in (A), 2 ␮m; (B), 0.5 ␮m; (C), 0.3 ␮m; (D), 1 ␮m.

The ESRD kidney from patient # 8 (54 years old and vacuolar Hϩ-ATPase recognized by Western blotting a from the United Kingdom) was characterized by severe single band at 31 kD (Fig. 4, left lane). Upon equilibra- sclerosis, with the glomeruli being hyalinized or present- tion of these particles in linear sucrose gradients, the ing a thickened capsule, and diffuse tubular epithelial cell brush-border marker enzyme, aminopeptidase, the baso- degeneration or atrophy (Fig. 2H). In addition, several lateral marker enzyme, Naϩ/Kϩ-ATPase, and the lyso- concretions of different size were observed within atrophic somal membrane marker, LAMP1, showed a broad dis- tubular lumen or interstitium, corresponding to calcifi- tribution with a large peak at fractions # 6–9, # 7–10, and cations (Fig. 2 I and J). # 7–10, respectively, whereas ClC-5 was restricted to a narrow peak at fractions # 7–8 (Fig. 4, fractions # 1–14). Ultrastructural analyses Particles containing the 31 kD subunit of Hϩ-ATPase Electron microscopy survey could be performed on essentially codistributed with ClC-5 at fractions # 7–8, the samples from the four Japanese and two patients and further showed a minor shoulder at fraction # 9 from the United States, and three controls. Extensive corresponding to the peak of lysosomes. These data analysis of patient # 3, who had a complete deletion of would indicate that, in the human kidney cortex, the bulk CLCN5, showed normal PT cells, including a thick and ϩ of H -ATPase is associated with ClC-5 in endosomes. well-differentiated brush border, coated invaginations and numerous endosomes of various sizes in the apical Immunocytochemistry area, normal lysosomes, as well as mitochondria that were abundant and elongated, and well-differentiated The locations of well-characterized plasma membrane tight junctions (Fig. 3). Similar observations were made proteins were studied by immunohistochemistry using in all patients. Nonspecific findings in PT cells, that in- frozen sections from the four Japanese patients with cluded dilation of the endoplasmic reticulum and appar- Dent’s disease and four control biopsies. Preliminary experiments established the validity of immunostaining ently smaller subapical areas containing few endosomes, ϩ were also observed in control samples. and confirmed the apical distribution of H -ATPase, megalin, and aminopeptidase, as well as the basolateral ؉ Subcellular distribution of vacuolar H -ATPase in the distribution of the ␣1 subunit of Naϩ/Kϩ-ATPase in con- human kidney: Western blotting trol biopsies (Fig. 5). In particular, Hϩ-ATPase was lo- In postnuclear particles from the normal human kid- cated in the subapical area of PT, and in the apical pole ney cortex, antibodies against the 31 kD subunit of the of ␣-type intercalated cells (IC) within collecting ducts Moulin et al: The kidney in Dent’s disease 1291

Fig. 4. Subcellular distribution of the vacuo- .lar H؉-ATPase in the humen kidney cortex Western blotting analysis. Postnuclear particles of a human kidney cortex (MLP, left lane) were equilibrated in a linear sucrose gradient (densest fractions are # 1ϩ2, loading zone fractions are # 13ϩ14). Equal volumes of the indicated fractions were further analyzed by Western blotting for the indicated constit- uents. Particles containing the 31 kD vacuolar Hϩ-ATPase codistribute with ClC-5 at fractions # 7 and # 8 (**), and further showed a minor shoulder at fraction # 9 corresponding to the peak of lysosomes as indicated by LAMP1 expression.

Fig. 5. Immunostaining for plasma membrane proteins in control renal biopsies. Immunostaining for the 31 kD subunit of vacuolar Hϩ-ATPase (A) conﬁrms its subapical distribution in proximal tubule (PT) cells as well as in the apical pole of ␣-type intercalated cells (arrowheads). Megalin (B) and aminopeptidase (C) are also located in the apical pole of PT epithelial cells, whereas the ␣1 subunit of the Naϩ/Kϩ-ATPase is located at the basolateral domain of the cells (D). Bar is 25 ␮m. 1292 Moulin et al: The kidney in Dent’s disease

Fig. 6. Immunostaining for plasma membrane proteins in the kidneys of patients with Dent’s disease. Serial sections (A to D) from patient # 3 with a deletion encompassing the entire CLCN5 gene. Immunostaining shows a complete inversion of polarity for Hϩ-ATPase in proximal tubule (PT) cells (A), contrasting with the preserved apical polarity of megalin (B) and aminopeptidase (C), and the preserved polarity of Naϩ/Kϩ- ATPase at the basolateral side of the cell (D). Adjacent sections from patient # 1 (E and F) and patient # 2 (G to H) confirm the inversion of polarity of Hϩ-ATPase (E and G) contrasting with the preserved apical polarity of megalin (F and H). Immunostaining for the anion exchanger AE1 and Hϩ-ATPase in patient # 3 (I and J) and patient # 2 (K and L). The basolateral staining for AE1 identifies ␣-type intercalated cells (*) within collecting duct profiles (I and K). No apical staining for Hϩ-ATPase can be detected in these cells (J and L) (*), whereas the basolateral staining in adjacent PT cells is preserved (L) (arrowheads). Bar is 40 ␮m.

(Fig. 5A). The distribution of these markers was similar tion of Hϩ-ATPase in PT cells (Fig. 6A), similar to that in all control biopsies examined. However, a similar anal- of Naϩ/Kϩ-ATPase (Fig. 6D), and this contrasted with ysis of the serial sections from Japanese patient # 3, the preserved apical distribution of megalin (Fig. 6B) characterized by a complete deletion of CLCN5, re- and aminopeptidase (Fig. 6C), as well as the apical polar- vealed unexpected ﬁndings. Immunostaining in this pa- ization of NHE3 and the mixed apical and basolateral tient with Dent’s disease revealed a basolateral distribu- distribution of the water channel AQP-1 (data not shown). Moulin et al: The kidney in Dent’s disease 1293

The selective inversion of the apical polarity of Hϩ- uria, mild phosphaturia, glycosuria, and polyuria [12, 13]. ATPase was confirmed in the four patients analyzed, in- Such PT dysfunction, when it occurs as a primary renal cluding patient # 1 (C-terminal deletion; Fig. 6 E and F), Fanconi syndrome in humans, may be associated with a patient#4(Tdeletion at 483; Fig. 6 G and H) and patient partial loss or shortening of the brush border [27]. The # 2 (not shown). The prolonged fixation times and areas absence of structural and ultrastructural changes in the of extended tubular damage did not allow us to perform PT cells of patients with Dent’s disease and ClC-5 knock- such reliable immunostaining experiments in the paraf- out mice is therefore unusual. The ClC-5 knockout mice fin-embedded samples from patients#6and#7. have been studied at a relatively young age [13], and it In addition to PT cells, ClC-5 and Hϩ-ATPase are is possible that subtle alterations in the endocytic appara- known also to colocalize in ␣-type IC, which can be tus of PT cells will appear with aging. However, this is independently identified by basolateral labeling of the unlikely to be the explanation in patients # 1 and # 6, who anion exchanger AE1 [23]. Strong basolateral staining were 14 and 31 years of age, respectively (Table 1). One for AE1 identified ␣-type IC in the collecting ducts of alternative possibility for the lack of morphologic alter- patients # 3 (Fig. 6I) and # 2 (Fig. 6K). However, these ations in the endocytic apparatus could be that the loss cells showed no apical staining for Hϩ-ATPase, which of ClC-5 does not affect the expression level of basic com- could nevertheless be clearly detected at the basolateral ponents of the endocytic apparatus such as clathrin and pole of adjacent PT cells (Fig. 6 J and L). Rab5 [10, 11], but specifically alters components that close- ly codistribute and perhaps physically interact with it. Our observations of Hϩ-ATPase distribution in the DISCUSSION human kidney (normal versus Dent’s disease) are consis- This study provides the first description of renal archi- tent with the latter hypothesis. In the normal human tecture in patients with Dent’s disease that have proven kidney, the vacuolar Hϩ-ATPase colocalizes with ClC-5 ClC-5 mutations. At the light microscope, the kidneys in the apical membrane area of PT cells as well as in ␣-type are characterized by hyaline casts and early calcifications. IC of the collecting ducts [7]. Furthermore, immunoblot Despite generalized PT dysfunction and LMWP, the ul- analyses revealed that Hϩ-ATPase codistributes with trastructure of PT cells is surprisingly normal, including ClC-5 in subcellular fractions compatible with endo- their tight junctions, brush border, and apical areas, that somes (Fig. 4). Dent’s disease, which is caused by a loss contain the endocytic apparatus. However, molecular ab- of functional ClC-5, is reflected by an inverted polarity ϩ normalities are detected in cells coexpressing H -ATPase of Hϩ-ATPase in PT cells and its apparent loss in the ϩ and ClC-5. There is a specific inversion of H -ATPase ␣-type IC (Fig. 6). These changes are specific for the polarity in PT cells and a loss of apical expression of vacuolar Hϩ-ATPase, as the polarity of other plasma ϩ H -ATPase in the ␣-type IC of the collecting ducts. membrane proteins in the same cells remains unaltered. The light microscopy studies revealed progressive, yet Furthermore, these changes are independent of the type nonspecific lesions, that include glomerular hyalinosis, of mutation. For example, a complete CLCN5 deletion tubular cell degeneration or atrophy, and mild interstitial (patient # 3) and a limited loss of the 98 amino acids at the fibrosis, confirming earlier observations on isolated cases C-terminus (patient # 1) resulted in identical alterations. [1, 24]. However, in addition to these nonspecific find- These modifications in the polarity and/or expression ings, our studies show that these kidneys also have hya- of a nonmutated protein point to physical interaction line casts that are sometimes calcified (Fig. 2). The casts between the C-terminus of ClC-5 and Hϩ-ATPase, that were invariably detected, even at the early stage and in would be essential for proper targeting (e.g., within PT otherwise normal kidneys. Their location in the outer cells) or even stability (e.g., within IC) of Hϩ-ATPase. medulla, presumably in the loop of Henle, as well as In contrast with the alterations observed in the kidney their distinct tinctorial affinity, suggests that they may of patients with Dent’s disease, it is puzzling to note that represent the first manifestations of nephrocalcinosis the distribution and polarity of Hϩ-ATPase is apparently [25]. This process was undoubtly accelerated in some unaltered in knockout mice lacking ClC-5 [12, 13]. In patients, such that calcifications were identified in a 6-year- absence of technical differences (similar fixation proce- old patient (Fig. 2). dures and antibodies were used in both species), it is LMWP is the most consistent feature of patients with tempting to speculate that the species difference may Dent’s disease [1, 3]. It has been demonstrated in the reflect the complexity of the multisubunit vacuolar Hϩ- two ClC-5 knockout mice [12, 13], but not in a ribozyme ATPase [28]. The complete identity of all the compo- antisense mouse model of reduced ClC-5 expression [26]. nents of the Hϩ-ATPase has yet to be elucidated, but The LMWP reflects a severe impairment in receptor- there are at least 13 subunits that present tissue- and mediated endocytosis in PT cells due to a loss of ClC-5 segment-specific expression [28]. For instance, different function. The impaired endocytosis is paralleled by other expression levels and segmental distribution of the manifestations of PT dysfunction, including aminoacid- 116 kD accessory (a) subunit isoforms have been docu- 1294 Moulin et al: The kidney in Dent’s disease mented in mouse and humans [29]. These differences ACKNOWLEDGMENTS could explain a different polarization of the 31 kD sub- This work was funded in part by IUAP, the Fondation Alphonse unit investigated here. Also, binding partners of the vacu- et Jean Forton, the Belgian agencies FNRS and FRSM, and Actions olar Hϩ-ATPase and ClC-5 in the kidney might be differ- de Recherches Concerteés (to O.D. and P.J.C.). R.V.T. is supported by the Wellcome Trust and MRC (UK). ent in humans and mouse. Finally, the discrepancy in The dedicated help of Y. Cnops, M. Leruth, L. Tanh, and L. Wender- polarization may reflect more general species differences ickx is acknowledged. We are indebted to the reviewers for their suggestions, and to Professors R. Beauwens (ULB Medical School, in nephron architecture, PT function, and/or rate of en- Brussels, Belgium), E.I. Christensen (University of Aarhus, Aarhus, docytosis [30, 31]. Denmark), and O. Wrong (UCL Medical School, London, UK) for The putative interaction between ClC-5 and Hϩ- additional material and fruitful discussions. ATPase would be of particular significance in PT cells, Reprint requests to Olivier Devuyst, M.D., Ph.D., Division of Ne- since stoichiometric fluxes of Hϩ and ClϪ are necessary phrology, UCL Medical School 10, Avenue Hippocrate, B-1200 Brus- to mediate endosomal acidification, which is a prerequi- sels, Belgium. E-mail: [email protected] site for progression along the endocytic apparatus [11]. Indeed, apical and basolateral targeting are extremely REFERENCES sensitive to mutations, and it has been shown that point 1. 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