Urinary Megalin Deficiency Implicates Abnormal Tubular Endocytic Function in Fanconi Syndrome

J Am Soc Nephrol 13: 125–133, 2002 Urinary Megalin Deficiency Implicates Abnormal Tubular Endocytic Function in Fanconi Syndrome

ANTHONY G. W. NORDEN,* MARTA LAPSLEY,† TAKASHI IGARASHI,‡ CATHERINE L. KELLEHER,§ PHILIP J. LEE,࿣ TAKESHI MATSUYAMA,¶ STEVEN J. SCHEINMAN,# HIROSHI SHIRAGA,** DAVID P. SUNDIN,†† RAJESH V. THAKKER,‡‡ ROBERT J. UNWIN,§§ PIERRE VERROUST,࿣࿣ and SØREN K. MOESTRUP¶¶ *Department of Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge, United Kingdom; †Department of Chemical Pathology, Epsom and St. Helier Trust, Epsom, United Kingdom; ‡Department of Pediatrics, Faculty of Medicine, University of Tokyo, Tokyo, Japan; §Division on Aging and Department of Genetics, Harvard Medical School, Boston, Massachusetts; ࿣Charles Dent Metabolic Unit and §§Centre for Nephrology, University College London Hospitals, London, United Kingdom; ¶Department of Pediatrics, Fussa Hospital, Tokyo, Japan; #Department of Medicine, State University of New York, Syracuse, New York; **Department of Paediatric Nephrology, Tokyo Women’s Medical University, Tokyo, Japan; ††Division of Nephrology, Indianapolis School of Medicine, Indianapolis, Indiana; ‡‡Molecular Endocrinology Group, Nuffield Department of Medicine, John Radcliffe Hospital, Oxford University, Oxford, United Kingdom; ࿣࿣INSERM U 538, Paris, France; and ¶¶Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.

Abstract. Normal reabsorption of glomerular filtrate pro- pixels and expressed as percentages of the normal mean teins probably requires recycling of the endocytic receptors values. A striking deficiency of urinary megalin, compared megalin (gp330) and cubilin. Both receptors are located on with normal individuals (n ϭ 42), was observed for eight of the luminal surface of the renal proximal tubule epithelium. nine families with Dent’s disease (n ϭ 10) and for the two Whether abnormal amounts of receptor are present in the families with Lowe’s syndrome (n ϭ 3). The family with urine of patients with Dent’s disease, Lowe’s syndrome, or autosomal dominant idiopathic Fanconi syndrome (n ϭ 2) autosomal dominant idiopathic Fanconi syndrome was ex- exhibited megalin levels within the normal range. The mea- plored. They are all forms of the renal Fanconi syndrome sured levels of cubilin were normal for all patients. These and are associated with tubular proteinuria. Urine samples results are consistent with defective recycling of megalin to of equal creatinine contents were dialyzed, lyophilized, and the apical cell surface of the proximal tubules and thus subjected to electrophoresis on nonreducing sodium dodecyl decreased loss into urine in Dent’s disease and Lowe’s sulfate-5% polyacrylamide gels. Proteins were blotted and syndrome. This defect would interfere with the normal probed with anti-megalin IgG, anti-cubilin IgG, or receptor- endocytic function of megalin, result in losses of potential associated protein. Megalin and cubilin levels detected by ligands into the urine, and produce tubular proteinuria. immunochemiluminescence were measured as integrated

The endocytic receptors megalin (gp330) and cubilin are ex- transmembrane domain but is thought to traffic with megalin pressed in high concentrations in renal proximal tubules, where on the cell surface (1,4). Mice in which the megalin gene has they mediate the uptake of a wide variety of protein ligands been “knocked out” excrete a variety of plasma proteins in from the glomerular filtrate (1–4). Megalin, which was origi- increased quantities in their urine, which provides further ev- nally identified in proximal tubule cells by Kerjaschki and idence for the importance of megalin-dependent pathways for Farquhar (5), is a type 1 integral membrane protein with 36 the recovery of low-molecular weight proteins and associated putative extracellular “ligand-binding repeats” and only a small vitamins from the glomerular filtrate (6). Megalin and cubilin cytoplasmic domain (1,5). In contrast, cubilin lacks a classic are recycled between apical clathrin-coated pits and early and late endosomes, with delivery of luminal ligands to the lyso- Received July 21, 2001. Accepted August 23, 2001. somal compartment, where they are hydrolyzed to component Correspondence to Dr. Anthony G. W. Norden, Department of Clinical Bio- amino acids. The details of this process remain to be eluci- chemistry, Box 232, Addenbrooke’s Hospital, Hill’s Road, Cambridge CB2 dated. In addition, megalin was recently found to mediate 2QR, UK. Phone: ϩ44-(0)1223-217609; Fax: ϩ44-(0)1223-216862; E-mail: [email protected] protein transcytosis in an immortalized rat cell line (7). As well as membrane-bound forms in tissues, both megalin 1046-6673/1301-0125 Journal of the American Society of Nephrology and cubilin have been detected in soluble form in human urine Copyright © 2001 by the American Society of Nephrology (8–11). At least some of the soluble megalin is a truncated 126 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 125–133, 2002 form of that associated with the renal brush border membrane syndrome, and ADIF syndrome. With the use of a validated (10). Similarly, rat proximal tubule cells in culture may pro- urine preparation method, megalin was observed to be almost duce both membrane-bound and soluble megalin (12). absent from the urine of most patients with Dent’s disease or The renal Fanconi syndrome (Lignac-de Toni-Debre´-Fan- Lowe’s syndrome. coni syndrome) consists of a generalized dysfunction of renal proximal tubules that leads, in its full form, to impaired proximal reabsorption of protein (tubular proteinuria), amino acids, Materials and Methods glucose, phosphate, urate, and bicarbonate and rickets/osteo- Patients malacia (13). Dent’s disease (CLCN5 mutation) (14,15), the A total of 13 affected male patients with Dent’s disease were oculocerebrorenal syndrome of Lowe (OCRL1 mutation) (16), studied. Each patient, unless otherwise stated, bore the CLCN5 mu- and autosomal dominant idiopathic Fanconi (ADIF) syndrome tation of the disease and exhibited characteristic clinical and labora- (17) are examples of renal Fanconi syndromes. Although other tory features, including tubular proteinuria (22–24). Details of the CLCN5 mutations and references to the full clinical descriptions and features of the Fanconi syndrome vary in these disorders, there laboratory findings for each patient are as follows: patients C/II/2 and is a consistent defect in renal tubular protein reabsorption (a C/III/2 (total CLCN5 deletion) (family 1) (22), patient F/II/1 (W279X process localized to the proximal tubules) (14). In Dent’s mutation) (family 2) (22), a member of family 7.1/94 (splice-site disease, in which a CLCN5 mutation leads to a defective mutation with deletion of codons 132 to 241) (family 3) (15), patient CLC-5 chloride channel, there is evidence of a failure to 4/96 (R34X mutation) (family 4) (25), patient 6/97 (346-amino acid acidify part of the endosomal compartment of proximal tubule deletion) (family 5) (26), two members of a family with a Ser244Leu cells (18–21). Furthermore, reduced apical expression of mutation (family 6) (24), one patient with an entire CLCN5 deletion megalin in the proximal tubules was recently demonstrated in (family 7) (26), and one patient with a TGG to TAG nonsense a mouse model of Dent’s disease (18). mutation at codon 343 (family 8) (26). Three patients from a family Defective recycling of megalin and cubilin receptors, with a GGG to GAG missense mutation at codon 506 (family 9) were whether attributable to the probable failure of endosomal acid- also studied. Two members of that family have mild atypical Dent’s disease, and one of those atypical patients (patient V-4) was one of the ification (as in Dent’s disease) or to other mechanisms, might three patients from family 9 studied here (15,27,28); the other two be expected to substantially change the rate of receptor trans- affected male subjects studied exhibit the typical features of Dent’s port to the luminal membrane, from which urinary receptors disease. The three patients with Lowe’s syndrome (all male) exhibit are presumably derived. We examined this hypothesis by severe mental retardation, growth retardation, visual impairment, and studying the urinary excretion of receptors in several forms of other clinical features typical of the disease, as well as tubular pro- the renal Fanconi syndrome, including Dent’s disease, Lowe’s teinuria (13,16); two of the patients are brothers. The two patients

Table 1. Urinary excretion of megalin and cubilin by patients with Dent’s disease, Lowe’s syndrome, or autosomal dominant idiopathic Fanconi syndrome, compared with normal individualsa

Dent’s Disease Normal Lowe’s Autosomal Dominant ϭ b Syndrome Idiopathic Fanconi (n 42) Families 1 to 8 Family 9 (n ϭ 3) Syndrome (n ϭ 2) (n ϭ 10) (n ϭ 3)

Megalin (%) mean 100 7 106 6 150 range 40 to 172 0 to 11 82 to 150 3 to 9 120 to 180 Cubilin (%) mean 100 124 125 115 110 range 45 to 185 75 to 164 80 to 145 78 to 130 70 to 152 Creatinine clearance 80 to 125 55 (34 to 101) 84 and 18,c 179d 33 (26 to 37) 58 (34 to 82) (ml/min per 1.73 m2) Retinol-binding protein 0.0085 Ϯ 0.00045f 23.6 Ϯ 5.3f 22.4 and 16.1,c 0.0083d 85 (68 to 93) 13.6 (5.1 to 22.1) excretion (mg/mmol creatinine)e

a Urinary excretion of megalin and cubilin was quantified as the band intensity (as integrated pixels) for the main megalin or cubilin bands for patients or normal individuals, expressed as a percentage of the integrated pixel (IP) value for a normal reference preparation of ϫ urine included on each gel [(IPPatient/IPReference) 100]. Each result was calculated as the mean of determinations on duplicate gels. b Family 9 includes patients with both typical and atypical features of Dent’s disease ((28)). c Two patients with typical Dent’s disease. d Patient V-4, with atypical Dent’s disease, lacks tubular proteinuria but bears a CLCN5 mutation. Individual patients are described in the Materials and Methods section. e Determined as an indicator of tubular proteinuria ((44)). f Mean Ϯ SEM. J Am Soc Nephrol 13: 125–133, 2002 Megalin and Fanconi Syndrome 127 with ADIF syndrome are father and son from the family that was mM Tris-HCl, 0.5 M sodium chloride, 5 mM calcium chloride (pH originally reported (17). Table 1 presents measurements of creatinine 7.5) (TBSCa buffer), at room temperature for 2 h and at 4°C over- clearance based on 24-h urine collections or calculated by using the night. For kidneys, 1 ml of 62.5 mM Tris-HCl (pH 6.8) with 10% Cockroft-Gault method (29). SDS, 20% glycerol, and 0.006% bromphenol blue was added to the homogenate from 200 mg of kidney, prepared as described above, Specimen Collection with incubation as for urine; an additional 10-fold dilution with 62.5 Midstream random urine specimens were collected without preser- mM Tris-HCl (pH 6.8) with 5% SDS, 20% glycerol, and 0.006% vative, after at least 18 h of sexual inactivity. The samples were bromphenol blue was made before electrophoresis. refrigerated immediately for up to 4 h and then either frozen at Ϫ80°C or in dry ice for up to 2 wk before transfer to liquid nitrogen for up to Immunoblotting 1 yr before analysis. Specimens were thawed once only before After blocking, membranes were rinsed in TTBSCa buffer (TBSCa analysis. buffer containing 0.1% Tween 20) and probed by using one of the following protocols: (1) receptor-associated protein (RAP): 0.4 ␮g/ml Preparation of Urine rat RAP (62221; Progen Biotechnik, Heidelberg Germany) for 2 h, a A volume of well mixed urine equivalent to 25 ␮mol of creatinine 1:1000 dilution of rabbit anti-human RAP (61098; Progen Biotechnik) was dialyzed (D9277 tubing; Sigma-Aldrich, Dorset, UK) for4hat (32) for 1 h, washing, and then a 1:40,000 dilution of anti-rabbit 4°C, with vigorous stirring, against3Lof50mMammonium bicar- F(ab')2 conjugated to horseradish peroxidase (HRP) (NA9340; Am- bonate (Fluka 09830; Sigma-Aldrich), with one change after 1 h. This ersham-Pharmacia Biotech, Buckinghamshire, UK); (2) anti-megalin: procedure removed Ͼ98% of the sodium and chloride from the urine. a 1:20,000 dilution of sheep anti-rat megalin (33) and then a 1:60,000 The dialysate was then lyophilized and stored at Ϫ80°C for up to 1 mo dilution of anti-sheep F(ab) conjugated to HRP (1301977; Roche until analysis. Urine samples dialyzed and lyophilized in this way Diagnostics, East Sussex, UK); (3) anti-cubilin: a 1:40,000 dilution of demonstrated good recovery of megalin and cubilin. In contrast, rabbit anti-human cubilin (33) containing 0.2 mg/ml human IgG ultrafiltration concentrators could not be used to process urine; these (I4506; Sigma-Aldrich) and then a 1:40,000 dilution of anti-rabbit produced large losses of the megalin present in normal urine, when F(ab')2 conjugated to HRP; (4) anti-cytoplasmic tail of megalin: a normal urine was mixed with the megalin-deficient Fanconi syndrome 1:20,000 dilution of a rabbit antibody to the cytoplasmic tail of urine and then concentrated by ultrafiltration. Addition of protease megalin (designated 459) (34) and then a 1:40,000 dilution of anti- inhibitors did not abolish these losses (data not shown). rabbit F(ab')2 conjugated to HRP. RAP and antibodies were prepared in TTBSCa buffer with 1 mg/ml bovine albumin. Washes between Urine Ultracentrifugation steps were performed with TTBSCa buffer. Final development was with ECLϩ reagent (Amersham-Pharmacia Biotech, Buckingham- Well mixed urine, to which protease inhibitors (P8340; Sigma- shire, UK). Aldrich) had been added at a concentration of 20 ␮l/ml urine, was centrifuged at 105 ϫ g for1hat4°C, in 1.5-ml aliquots. Supernatants were pooled and processed by rapid dialysis and lyophilization as Chemiluminescence Recording described above. The pellets were resuspended in the same volume of Film (Bio-Max Light; Kodak, Hertfordshire, UK) was preflashed 50 mM ammonium bicarbonate as the original urine samples and were before exposure, and signals from scanned images (Epson GT-9500 dialyzed and lyophilized as described above. Image Scanner with EU-14 transmission mode unit; Epson UK, Hert- fordshire, UK) were processed with SigmaGel software (SPSS Inc., Preparation of Kidney Homogenates Chicago, IL) to yield an integrated pixel count for each band. Relative Normal human kidney cortex (200 mg wet weight) obtained during receptor excretion was then calculated as described in the footnote to partial nephrectomy was homogenized on ice, with a ground-glass Table 1 and the legend to Figure 2. homogenizer, in 0.8 ml of 62.5 mM Tris-HCl (pH 6.8) containing protease inhibitors (20 ␮l/ml, P8340; Sigma-Aldrich). Results When electroblots of SDS-polyacrylamide gels containing Megalin and Cubilin normal urine samples were probed with RAP, the binding Megalin and cubilin, purified as described (30,31), and prestained pattern was dominated by a single band, which comigrated molecular weight markers (C3312; Sigma-Aldrich) were used as with authentic 600-kD megalin (Figure 1A, lanes 5 and 6). standards for gel electrophoresis. RAP is a high-affinity, chaperone-like ligand for megalin (10). Using the RAP binding system, we compared normal urine Sodium Dodecyl Sulfate-Gel Electrophoresis and samples with urine specimens from patients with Dent’s dis- Blotting ease (Figure 1A, lanes 8 and 9), Lowe’s syndrome (Figure 1A, Urine lyophilizates were redissolved in 0.5 ml of 62.5 mM Tris- lanes 1 to 4), or ADIF syndrome (Figure 1A, lane 7). Figure 1A HCl (pH 6.8) with 5% sodium dodecyl sulfate (SDS), 10% glycerol, demonstrates a marked deficiency of all forms of megalin in and 0.003% bromphenol blue and were heated at 40°C for 30 min. the urine of the two patients with Dent’s disease and the three Twenty-five microliters were applied to 5% polyacrylamide gels patients with Lowe’s syndrome but not in the one study patient (161-1210; Bio-Rad, Hertfordshire, UK) and subjected to electrophoresis in 25 mM Tris, 0.192 M glycine, at 200 V for approximately with ADIF syndrome. To corroborate these results, we also 35 min. Proteins were transferred to polyvinylidene difluoride mem- detected the receptor by using a polyclonal antibody to mega- branes (Immobilon-P; Millipore, Hertfordshire, UK) in 25 mM Tris, lin, instead of RAP binding. As indicated in Figure 1B, iden- 0.192 M glycine, at 150 V (limited to 35-W maximal power). Blots tical results were obtained by using anti-megalin antibodies, were blocked with 3% bovine albumin (A7638; Sigma-Aldrich) in 20 compared with RAP binding. The results presented in Figure 1 128 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 125–133, 2002

Figure 1. Megalin deficiency in the urine of patients with Dent’s disease and Lowe’s syndrome but not autosomal dominant idiopathic Fanconi (ADIF) syndrome. Lanes 1 and 2, a patient with Lowe’s Figure 2. Urinary excretion of total megalin quantified for normal syndrome, in duplicate; lanes 3 and 4, two additional patients with individuals (n ϭ 42) and patients with Dent’s disease (families 1 to 8, Lowe’s syndrome; lanes 5 and 6, two normal male subjects; lane 7, a n ϭ 10; family 9, n ϭ 3), Lowe’s syndrome (n ϭ 3), and ADIF patient with ADIF syndrome; lanes 8 and 9, two patients with Dent’s syndrome (n ϭ 2). Band intensities (as integrated pixels) for the main disease. The images present the chemiluminescence of electroblots megalin or cubilin bands for patients or normal individuals were after urine was subjected to electrophoresis on 5% nonreducing so- expressed as a percentage of the integrated pixel (IP) value for a dium dodecyl sulfate (SDS) gels and megalin was detected by recep- normal reference preparation of urine included on each gel [(IPPatient/ ϫ tor-associated protein (RAP) binding (RAP followed by anti-RAP and IPReference) 100]. Each data point represents the mean of determi- peroxidase-conjugated, species-specific antibodies) (A) or anti-mega- nations on duplicate gels. Table 1 and the Materials and Methods lin antibody binding (anti-megalin and peroxidase-conjugated, spe- section provide details for each patient group. cies-specific antibodies) (B). The positions of native megalin (600 kD) and 220-kD, 116-kD, and 65-kD molecular mass standard are indicated by arrows. same range as values for patients with Dent’s disease or Lowe’s syndrome with megalin deficiency (Figure 2 and Table were confirmed with at least one more urine sample from each 1); therefore, the findings for these patients with Fanconi patient, obtained several weeks later. syndrome are unlikely to be attributable to normal variation. To explore this finding further, we examined urine speci- Among patients with Dent’s disease (families 1 to 8), Lowe’s mens from 11 more patients with Dent’s disease and one syndrome, or ADIF syndrome, we observed no significant additional patient with ADIF syndrome. The quantified nor- correlation between urinary megalin levels and either renal malized data for megalin excretion by all patients studied are function (as estimated on the basis of creatinine clearance) or presented in Figure 2 and Table 1. These data demonstrated the degree of low-molecular weight proteinuria (as assessed on almost complete deficiency of total urinary megalin for 10 the basis of retinol-binding protein excretion) (Table 1 and data patients with Dent’s disease, representing eight of the nine not shown). In the atypical family with Dent’s disease (family families studied. All three patients with Lowe’s syndrome were 9), without urinary megalin deficiency, the detected megalin deficient in total urinary megalin. For the two patients with was investigated further (see below). ADIF syndrome, no deficiency of either total or soluble mega- Cubilin is an endocytic receptor that exhibits ligand-binding lin was observed (Figure 2 and Table 1). None of the normal properties distinct from those of megalin but may interact with individuals whose urine was examined exhibited results in the megalin as a coreceptor (35,36). We therefore examined nor- J Am Soc Nephrol 13: 125–133, 2002 Megalin and Fanconi Syndrome 129

contrast, use of ultrafiltration concentrators led to losses of these receptors (data not shown). The standard method used for the preparation of receptors from urine was specifically intended to include all species of receptors, i.e., those that might be bound to cell membranes or adsorbed onto particulate material as well as unbound forms. The results obtained with this method are considered to represent total urinary megalin levels. In addition, we determined whether there were soluble forms of receptors in the urine of these patients, as well as forms that could be sedimented by ultracentrifugation. Using the ultracentrifugation technique described in the Materials and Methods section, we analyzed urine samples by immunoblotting. In normal urine, little megalin or cubilin could be sedimented by ultracentrifugation (Fig- ure 4, a and b), suggesting that both megalin and cubilin are present in urine primarily in a soluble form. In contrast, significant amounts of urinary megalin from the urine of a patient with typical Dent’s disease in family 9 were sedimented by ultracentrifugation (Figure 4c). Similar results were observed for one other member of family 9 with typical Dent’s disease Figure 3. Presence of cubilin in normal quantities in the urine of and also a family member (patient V-4) with atypical disease patients with Dent’s disease, Lowe’s syndrome, or ADIF syndrome. (data not shown). Negligible amounts of megalin could be Lanes 1 and 2, a patient with Lowe’s syndrome, in duplicate; lane 3, a second patient with Lowe’s syndrome; lane 4, a normal male sedimented by ultracentrifugation from the urine of the two subject; lane 5, a patient with Dent’s disease; lane 6, a patient with patients with ADIF syndrome (data not shown). ADIF syndrome; lane 7, a normal male subject (urine applied in To further characterize the megalin excreted in urine, we one-tenth the quantity of that in lane 4); lanes 8 and 9, two patients examined whether the megalin in normal urine would react with Dent’s disease. The image presents the chemiluminescence of an with the polyclonal antibody (antibody 459) (34) raised against electroblot after urine (prepared as described in the Materials and a sequence in the cytoplasmic tail of megalin. Using this Methods section) was subjected to electrophoresis on 5% nonreducing anti-cytoplasmic tail antibody, we observed no reactivity in a SDS-polyacrylamide gels and cubilin was detected by the binding of preparation from normal urine, under conditions in which a anti-cubilin and peroxidase-conjugated, species-specific antibodies. strong signal was observed in samples of human whole-kidney The migration positions of native cubilin (460 kD) and molecular homogenate. The megalin antibody that had been raised mass standards are indicated by arrows; the unlabeled arrow indicates against whole megalin yielded strong signals in both normal a probable cubilin dimer (see Discussion). urine preparations and human kidney homogenates (as controls). Similarly, there was no reactivity of antibody 459 with the form of megalin that was sedimented by ultracentrifugation mal urine samples and urine samples obtained from patients of urine from patients with Dent’s disease in family 9 (data not with the renal Fanconi syndrome for cubilin. In contrast to shown). megalin, no deficiency was observed (Figure 3 and Table 1). As controls for nonspecific interactions, the RAP binding Discussion immunoblots were examined with omission of RAP, incuba- These results are consistent with the involvement of defec- tion with nonimmune rabbit serum in place of anti-RAP, or tive trafficking of megalin in the pathogenesis of two major omission of anti-RAP. No significant bands corresponding to forms of the renal Fanconi syndrome, i.e., Dent’s disease and protein molecular masses of Ͼ180 kD were detected with Lowe’s syndrome (15,37), and are also consistent with the either normal urine or urine from a patient with Fanconi recent finding of reduced apical expression of megalin in the syndrome. Similarly, use of nonimmune serum in place of the renal proximal tubules in a mouse model of Dent’s disease first antibody or omission of the first antibody in the anti- (18). We suggest that failure to traffic megalin to the apical megalin system did not generate significant bands (data not epithelium in proximal tubules, as part of normal recycling, shown). results in a marked decrease in the loss of megalin into luminal To detect possible urine-induced proteolysis in the samples fluid and thus into urine (Figure 5). In vitro studies previously from patients with Dent’s disease or Lowe’s syndrome, we suggested a central role for megalin in the pathogenesis of this examined the stability of the receptors megalin and cubilin in disease (20,21). urine, by incubating together normal urine and megalin-defi- A large body of work has established megalin as a major cient urine from patients with Dent’s disease, for up to6hat endocytic receptor in proximal tubules that binds several fil- ␤ 37°C. No significant loss of megalin or cubilin was observed tered proteins, including 2-microglobulin, retinol-binding ␤ when the dialysis and lyophilization protocol described in the protein, vitamin D-binding protein, and 2-glycoprotein I Materials and Methods section was used to process urine. In (1,6,38). Cubilin, in association with megalin, has recently 130 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 125–133, 2002

Figure 5. Model of megalin trafficking in a proximal tubule cell, to account for the decreased loss of megalin into urine for patients with the renal Fanconi syndrome attributable to Dent’s disease. Normal acidification of the sorting endosomes requires activity of both the vacuolar Hϩ-ATPase (Hϩ 3) and the CLC-5 chloride channel (ClϪ 3), and there is failure of vesicle acidification in Dent’s disease because of the defective CLC-5 chloride channel. Normal recycling of megalin to the cell surface and physiologic release of the receptor therefore does not occur (see Discussion). Details of other known members of the endocytic apparatus, such as AP-2, are omitted. It is unclear whether acidification facilitated by CLC-5 has a direct effect on ligand dissociation from the receptor, as shown here, or an indirect effect attributable to failure to recruit endosomal regulatory proteins, as recently proposed by Maranda et al. (45).

signaling to the cell cytoplasm (40,41). Disruption of normal megalin trafficking would therefore be expected to interfere with both endocytic and signaling functions. It is unclear why family 9 differs from the eight other families with Dent’s disease in excreting significant amounts of urinary megalin (Figure 2 and Table 1). Furthermore, unlike normal urinary megalin, the megalin in the urine of patients Figure 4. Forms of megalin and cubilin in urine. (a and b) Megalin (a) and from this family occurs in a form that is sedimented by ultra- cubilin (b) are present mostly in soluble form in normal urine. When normal centrifugation (Figure 4). This family is also unusual in that urine was ultracentrifuged at 105 ϫ g for 1 h, as described in the Materials two male patients with a CLCN5 mutation have very mild, and Methods section, essentially all of the receptor present in the original atypical disease, with either undetectable or slight tubular urine sample (lane 2) was recovered in the supernatant (lane 4), with almost proteinuria, although the same mutation causes severe disease none in the pellet (lane 3). Authentic megalin and cubilin are shown in lane and marked tubular proteinuria among other members of the 1. The positions of native megalin (600 kD) and cubilin (460 kD) and a same family (28). The mutation in this family is a missense 220-kD molecular mass standard are indicated by thick arrows; the unlabeled mutation substituting glutamine for glycine at codon 506 and is arrow in b indicates a probable cubilin dimer (see Discussion). (c) Unlike predicted to disrupt charge distribution in the highly conserved findings for patients with Dent’s disease from families 1 to 8, megalin was 11th transmembrane domain of the channel protein. When not deficient in the urine of a patient from family 9 and, unlike that in normal expressed in Xenopus oocytes, the mutant CLC-5 produces a urine, megalin in the original urine sample from an affected patient with Dent’s disease in family 9 (lane 1) was present in both the pellet (lane 2) and chloride conductance that is indistinguishable from that in the supernatant (lane 3) after ultracentrifugation at 105 ϫ g for1h. uninjected control cells and is indistinguishable from that produced by other mutations (missense, nonsense, and other mutations), represented in this study by patients with absent been observed to be an albumin receptor in the kidney (35,36), urinary megalin (15). We speculate that different CLCN5 mu- in addition to its previously identified role as a receptor for tations can have different effects on CLC-5, which are associ- vitamin B12-intrinsic factor in the ileum (39). Megalin is also ated with the shedding of distinct forms of megalin into the thought to have roles in Ca2ϩ transport and sensing, as well as lumen of the proximal tubules and thence into urine. J Am Soc Nephrol 13: 125–133, 2002 Megalin and Fanconi Syndrome 131

Findings for the family with ADIF syndrome indicate that two major forms of the renal Fanconi syndrome, i.e., Dent’s the urinary megalin deficiency observed in Dent’s disease and disease and Lowe’s syndrome. This complements recent stud- Lowe’s syndrome is specific and not secondary to the presence ies of ligand binding and cellular and animal models. Figure 5 of other material such as proteases, which might be excreted in presents a model to account for our findings for patients with increased concentrations as part of the tubular proteinuria of Dent’s disease. On the basis of these studies, it may be possible the Fanconi syndrome. Furthermore, if the deficiency of mega- to use the detection of megalin in urine as a functional assay to lin were attributable to the presence of proteases, then pro- diagnose the failure or disruption of endocytosis and traffick- longed in vitro coincubation of normal urine containing mega- ing in renal proximal tubules. lin with urine from patients lacking urinary megalin should have resulted in megalin losses. This was not observed, which Acknowledgments further suggests that the observed deficiency of urinary mega- This work was supported by grants from the Sir Jules Thorn lin is not due to proteolytic or other events after shedding into Charitable Trust (to Dr. Norden), the National Institutes of Health the tubular lumen. (Grant DK46838, to Dr. Scheinman), and the Medical Research Lowe’s syndrome is attributable to mutation of the OCRL1 Council and Wellcome Trust (to Dr. Thakker). Part of this work was gene, which encodes a phosphatidylinositol-4,5-bisphosphate presented at the 33rd Annual Meeting of the American Society of phosphatase (37). The results presented here implicate abnor- Nephrology, October 13 to 16, 2000. We thank Prof. C. N. Hales for helpful discussions. mal megalin receptor-mediated endocytosis in Lowe’s syndrome. Phosphatidylinositol-4,5-bisphosphate is localized pri- References marily on the cytoplasmic face of the plasma membrane, and a 1. Christensen E, Birn H: Megalin and cubilin: Synergistic endo- role in the assembly of endocytic clathrin-coated vesicles has cytic receptors in renal proximal tubule. Am J Physiol 280: been identified (42). However, a link between the phosphati- F562–F573, 2001 dylinositol-4,5-bisphosphate phosphatase defect and the possi- 2. Orlando RA, Rader K, Authier F, Yamazaki H, Posner BI, ble endocytic defect in this syndrome remains to be described. 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