The urinary proteome in Fanconi syndrome implies specificity in the reabsorption - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Pedro R. Cutillas,,, Robert J. Chalkley, Kirk C. Hansen, Rainer Cramer,, Anthony G. W. Norden, Mike D. Waterfield,, Alma L. Burlingame, and Robert J. Unwin作者单位：1 Ludwig Institute for Cancer Research, London W1W 7BS; 2 Department of Biochemistry and Molecular Biology and 3 Centre for Nephrology and Department of Physiology, Royal Free and University College Medical School, University College London, London NW3 2PF; 5 Department of Clinical Biochemistry, Add ; Z9 u, `/ l, n* ~/ b% L! M/ G
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      【摘要】- t( S, }* h7 _
   Polypeptides present in the glomerular filtrate are almost completely reabsorbed in the first segment of the proximal tubule by receptor-mediated endocytosis; in renal Fanconi syndrome (FS), there is failure to reabsorb many of these polypeptides. We have compared the urinary proteomes in patients with Dents disease (due to a CLC5 mutation), a form of FS, with normal subjects using three different proteomic methods. No differences in the levels of several plasma proteins were detected when standardized to total protein amounts. In contrast, several vitamin and prosthetic group carrier proteins were found in higher amounts in Dents urine (with respect to total protein). Similarly, complement components, apolipoproteins, and some cytokines represented a larger proportion of the Dents urinary proteome, suggesting that such proteins are reabsorbed more efficiently than other classes of proteins. Conversely, proteins of renal origin were found in proportionately higher amounts in normal urine. Thus the uptake of filtered vitamins, which are normally bound to their respective carrier proteins to prevent urinary losses, seems a key function of the proximal tubule; in addition, this nephron segment may also play a critical role in reabsorbing potentially cytotoxic polypeptides of plasma origin, preventing them from acting at more distal nephron sites.
      【关键词】 kidney urine proteomics mass spectroscopy4 b0 q6 G! q0 _9 D) ^; [# e
   A RECEPTOR - MEDIATED ENDOCYTIC process in the first part of the proximal tubule reabsorbs small- and medium-sized polypeptides, which are freely filtered at the kidney glomerulus. This pathway is important for the salvage of amino acids in the form of protein ( 1, 8 ), vitamins (bound to their carrier proteins) ( 3, 6, 28, 38, 47 ), and hormones ( 19, 31, 42 ). Major receptors are megalin and cubilin, which act synergistically as scavenger receptors and appear to mediate the reuptake of almost all filtered polypeptides ( 4 ). After internalization of the megalin-cubilin-ligand complex within early endosomes, a vacuolar electrogenic H -ATPase pump acidifies them and facilitates dissociation of the receptor-ligand complex; the receptor is then recycled to the plasma membrane and the ligand is taken up and degraded by lysosomes. The chloride channel ClC-5 has been shown to be critical in this process, because it allows chloride entry into endosomes, which shunt the electrical gradient generated by the vacuolar H -ATPase ( 13 ). As a consequence of the loss of function of ClC-5, recycling of megalin to the plasma membrane is probably disrupted, which, in turn, affects megalin-mediated endocytosis of polypeptides ( 5 ). Proteins filtered by the glomerulus are almost completely removed from tubular fluid by this process, such that none or only trace amounts normally appear in urine.) I# g3 r! I$ l
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Mice lacking functional ClC-5 have many of the features of renal Fanconi syndrome ( 43, 50 ), a hallmark of which is low-molecular-mass (tubular) proteinuria. Dents disease is the clinical form of renal Fanconi syndrome due to loss-of-function mutations of CLCN5 on the X chromosome ( 29 ). In addition to tubular proteinuria, manifestations of Dents disease include nephrolithiasis, hypercalciuria, nephrocalcinosis, and in the majority of cases, progressive renal failure ( 52 ).1 P1 t6 U8 R6 }) {5 S
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Although the presence of abnormally high concentrations of proteins and peptides in the urine of CLCN5 knockout mice and Dents disease patients is now well documented ( 9, 36, 37, 43, 50 ), there are no detailed reports on the relative abundance and/or qualitative protein composition of the urine of the mouse model or of patients. Here, we present the results of a study in which we have made a qualitative comparison, and an assessment of relative abundance, between the urinary proteomes of Dents disease patients and normal subjects. Three different comparative proteomic strategies were used to enhance the number and confidence of protein identifications. In every case, the analysis was standardized to protein total amounts. Two-dimensional gel electrophoresis (2DE) was first used to compare the patterns of Dents and control (healthy subjects with normal renal function) urinary proteomes. The identities of the protein spots were determined by peptide mass mapping (PMM) using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS). In a parallel investigation, proteins separated by microcapillary liquid chromatography (µLC) were digested with a protease; the resulting peptide products were then sequenced by online liquid chromatography tandem mass spectrometry (LC-MS/MS). Finally, the results of these two different methods of analysis were validated using a relatively new analytic approach known as isotope-coded affinity tags (ICAT): Dents disease and control urinary proteins were labeled with heavy and light stable isotopes, respectively. After the samples were mixed, multidimensional LC-MS/MS was then used to identify and accurately quantitate the fold-differences between Dents and normal urinary proteins.
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The results obtained by these three different analytic methods were highly consistent. We detected several vitamin and prosthetic group carrier proteins, apolipoproteins, complement components, and potentially bioactive peptides, all at higher levels (that is, the amount of protein normalized to total protein) in Dents urine compared with normal urine. These findings strongly suggest that such proteins are reabsorbed from the glomerular filtrate in preference to other classes of protein.

METHODS
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Subjects. We studied four male patients with X-linked Dents disease, all with well-characterized mutations of the CLCN5 gene and whose phenotypic and genotypic features have been described previously in detail elsewhere ( 7, 29, 52, 53 ). Their ages were 18, 29, 38, and 44 yr, with corresponding estimates (using the MDRD 0.84 correction factor applied to the modified Cockcroft-Gault formula for estimating creatinine clearance) ( 26 ) of glomerular 80), 47, and 26 ml/min, respectively. All had significant tubular proteinuria (measured as retinol binding protein). Control urine (designated as "normal") was collected from five male subjects with no history of renal disease and ages ranging between 25 and 35 yr. Patients and volunteer subjects gave informed consent for the collection and analysis of their urine. After collection, urine samples were either processed immediately or frozen in liquid nitrogen and stored at -80°C. On the day of analysis, all samples were prepared so as to yield sample solutions with final concentrations of 1 mM EDTA, 1 µg/ml aprotinin, and 0.1% trifluoroacetic acid (TFA).
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Gel electrophoresis. Proteins were desalted using a column packed in house with reverse-phase (RP) material (C 18, POROS R2 20, 2-ml bed volume, Applied Biosystems, Warrington, UK) as reported previously ( 9 ). Protein concentrations were then quantified by the Bradford assay. 1DE (SDS-PAGE) was carried out using 12 and 15% bis-acrylamide gels. 2DE was carried out as described previously ( 11 ) in 18-cm IPG strips (Amersham Biosciences, Amersham, UK). For the second dimension, we used 10, 12, and 15% SDS-PAGE gels run in triplicate. Gels were scanned using a densitometer (Bio-Rad 800), and gel images were analyzed using Melanie 3.8 software, which allowed spot matching and quantitation. Spot intensity was expressed as the percentage of optical density (OD) relative to total OD. Proteins were visualized using a mass spectrometry-compatible silver staining procedure ( 46 ). Proteins present in gel bands or gel spots were prepared for mass spectrometric analysis by in-gel digestion following the procedure of Shevchenko et al. ( 46 ) with modifications described elsewhere ( 2 ).

Microcapillary HPLC. Polypeptides were desalted as for gel electrophoresis and further extracted using a SCX column (POROS HS 20, 800-µm ID x 50-mm long) as described previously ( 9 ). Protein concentrations were then quantified using the Bradford assay and subsequently separated by µLC using a RP capillary column (POROS 10 R2, 320-µm ID x 250-mm long) packed in-house as described elsewhere ( 45 ). Gradient elution was carried out from 15% B to 60% B in 30 min, after an initial isocratic step of 8 min at 5% B. Mobile phase A was 0.1% TFA, and mobile phase B was 80% ACN/0.1% TFA. The flow rate was 20 µl/min, and fractions were collected every 3 min; the proteins present in each fraction were digested with trypsin overnight in 50 mM ammonium bicarbonate at pH 8.5.' n- ]5 L5 n# l$ o3 ?5 u2 i
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Labeling of proteins with isotope-coded affinity tag reagents. Before isotopic labeling, proteins were precipitated in 50% acetone and further extracted by SCX as above. Dents disease proteins were labeled with the heavy cleavable ICAT reagent (Applied Biosystems, Framingham, MA), whereas normal urinary proteins were labeled with the light ICAT reagent as recommended by the manufacturer with the modifications as described previously ( 17 ). Briefly, proteins were resuspended in 6 M urea/50 mM ammonium bicarbonate/5 mM Tris(2-carboxyethyl)phosphine and subsequently labeled with the cleavable ICAT reagents for 2 h at 37°C in the dark. A 50-µg aliquot of each Dents urine sample was individually mixed with an equal amount of a pool of five normal urinary proteins, adjusted to 1 M urea and with 10 µg of sequencing grade trypsin added to the reaction mixture, which was then incubated overnight at 37°C. ICAT-labeled peptides were separated and analyzed as described elsewhere ( 17 ), with the exception that the nonlabeled peptides were not analyzed and the fractions containing ICAT-labeled peptides (4 fractions/sample) were not pooled.
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MS. The identities of the proteins in the gel spots or chromatographic fractions were determined by MALDI-MS and/or LC-electrospray ionization (ESI)-MS/MS. MALDI mass spectra of 2DE spots were acquired using an Ultraflex TOF/TOF instrument (Bruker Daltonics, Bremen, Germany). LC-ESI-MS/MS was performed using an Ultimate nanoflow HPLC system (LC Packings, Amsterdam, The Netherlands) connected online to a Q-Tof mass spectrometer (Micromass, Manchester, UK) or to a QSTAR instrument (MDS-Sciex, Toronto, Ontario). ICAT-labeled peptides were also analyzed by LC-MALDI-MS/MS using a 4700 Proteomics Analyzer (Applied Biosystems). Details of the operation and settings of these instruments, as well data analysis, have already been described in detail elsewhere ( 2, 9, 11, 17 ).
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RESULTS- j7 ]  t/ V0 Q; A6 ~7 a

Analysis of urinary polypeptides by gel electrophoresis. The protein concentration in Dents urine is 30-60 times higher than in the urine of normal subjects ( 36, 37 ). However, because we were interested in the qualitative, rather than the quantitative, differences between Dents and control urinary proteomes, the same numbers of Dents disease and normal urinary proteins were separated by SDS-PAGE ( Fig. 1 ). No bands were visible above 70 kDa for the Dents proteins. In contrast, four major bands above this molecular mass were present in the normal samples. The identities of the proteins present in three of these bands were determined by LC-ESI-MS/MS and indicated that megalin, cubilin, epidermal growth factor (EGF) precursor, and uromodulin represent a larger proportion of the normal urinary proteome compared with the Dents proteome. These proteins are all known to be of renal origin, and their low relative concentration in Dents urine with respect to total protein may be due the large amount of protein derived from plasma that is present in Dents urine. The identities of the other bands present in the gel image shown in Fig. 1 were not determined.
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Fig. 1. SDS-PAGE separation of Dents and normal urinary proteins. Urinary proteins of 4 normal and 4 Dents disease subjects were separated by SDS-PAGE and stained with silver nitrate. Fifty micrograms of protein were loaded in each lane. The identities of the proteins present in bands ( 1-4 ) were determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The no. of peptides with derived sequence information and the percentage of protein sequenced by MS/MS are listed. NCBI, National Center for Biotechnology Information.
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We also compared the protein patterns present in the urine of Dents patients with normal individuals by 2DE ( Fig. 2 ). Gels with three different acrylamide/bis-acrylamide concentrations (10, 12, and 15%) were used in preliminary experiments. Spot patterns between these gels were largely consistent; however, for simplicity, only the 15% gels were subjected to further analysis. Equal amounts of protein (150 µg) from normal and Dents urines were loaded. After electrophoretic separation and silver nitrate staining, 192 spots were excised from each of the 2 gels displayed in Fig. 2, and the identities of the proteins found in 174 of these spots were determined by MS, resulting in 50 different gene products ( Tables 1 - 3 ). Several proteins were found to be at higher levels in the urine of Dents disease patients compared with normal urine, including the spots corresponding to apolipoprotein AI (apo-AI, spots 3-5 in Fig. 2 A ) and apolipoprotein AIV (apo-AIV, spots 6-8 ), hemopexin (HPX), pigment epithelium-derived factor (PEDF), retinol binding protein (RBP), transthyretin (TTR), and vitamin D binding protein (VDBP). Proteins found at higher levels in normal urine than in Dents urine also included kininogen ( spots 42, 93, 115, and 123-143 ) and uromodulin ( spots 96-98 ), which occupied a relatively large percentage of the normal urinary proteome but were not found in Dents urine by 2DE. Finally, there were proteins present at similar levels in normal urine and the urine of Dents patients. Most of these proteins are (abundant) plasma proteins like albumin ( spots 47-69 ), microglobulins ( spots 96-98 ), and immunoglobulins ( spots 102-109 ).

Fig. 2. Two-dimensional gel electrophoretic profiles of Dents and normal urinary proteomes. A : representative gel images of the proteome of Dents disease urine ( left ) and normal urine ( right ) are shown. Same amounts of protein (150 µg) were loaded on each gel and detected by silver staining. Identities of the labeled spots were determined by mass spectrometry as described in METHODS. Spot numbers correspond to those in Tables 1 - 3. B : selected areas of the gels in A showing differential spot patterns are enlarged. Dents disease proteins are shown on the left and normal proteins on the right. Arrows point to the electrophoretic migration position of the named gene product. PEDF, pigment epithelium-derived factor; HPX, hemopexin; RBP, retinol binding protein; VDBP, vitamin D binding protein; TTR, transthyretin.

Table 1. Proteins found at higher levels (amounts relative to total protein) in Dents than in normal urine by 2DE6 Q$ `: L" h6 T5 N; t5 \; C

Table 3. Proteins found at higher levels (relative to total protein) in normal than in Dents urine by 2DE8 Q: g/ L. k7 a: Y

Analysis of urinary polypeptides by µLC and LC-ESI-MS/MS. To investigate whether the differences observed by 2DE could also be detected by a different analytic approach, equal amounts (5 µg) of Dents and normal urinary proteins were also separated by RP µLC and analyzed by LC-ESI-MS/MS. This approach identified 124 polypeptides, which are listed in Table 4. Relative protein amounts between the two sample groups were compared on the basis of peak ion intensities. As in the 2DE analysis, results showed that HPX, 2 -GP I, VDBP, TTR, and RBP were present at higher levels in the urine of Dents patients, whereas tryptic peptides of uromodulin produced higher ion intensities in normal urine ( Fig. 3 ). In addition, Fig. 3 also shows that several apolipoproteins were present at higher levels in the urine of Dents patients compared with the urine of normal individuals, except for apo-D, which occurred at higher levels in normal urine. Finally, polypeptides with a potential signaling function were also detected. In agreement with the 2DE data, kininogen peptides produced stronger ion intensities in the normal samples, whereas PEDF tryptic peptides were found in Dents urine but not in normal urine. Other potentially bioactive peptides not seen in the 2DE analysis were found in Dents urine by µLC but not in normal urine ( Fig. 3 ). These included proplatelet basic peptide (PBP), IGF-II, diazepam binding inhibitor (DBI), angiomodulin (AGM), PDGF, and bone morphogenic protein-1. In contrast, osteopontin (OPN) produced stronger ion signals in normal urine.

Table 4. Proteins identified by LC-MS/MS from the µLC fractions4 q7 S* ]! _: f
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Fig. 3. LC-MS elution profile of tryptic peptides from urinary proteins. Polypeptides (5 µg) purified from urine by reverse-phase (RP) followed by SCX chromatography were loaded on a microcapillary liquid chromatography (µLC) system as described in METHODS. µLC runs were carried out in triplicate for 4 patients and 4 healthy subjects, and the elution profiles (as determined by UV absorption) were highly consistent within sample groups (data not shown). Ten fractions were collected every 3 min, lyophilized, and the proteins were digested with trypsin. LC-ESI-MS/MS was then used to determine the identity of the gene products in each fraction. Extracted ion chromatograms of the precursor peptide ions are displayed together with their intensities. The elution peak of the corresponding peptide ion is indicated by an arrowhead. Note that in some cases more than 1 ion had the same mass/charge ratio ( m / z ). OPN, osteopontin; PBP, proplatelet basic peptide; DBI, diazepam binding inhibitor; AGM, angiomodulin; UMOD, uromodulin; IGFBP-7, insulin-like growth factor binding protein-7; Apo, apolipoprotein.) u( Z6 F- k* s6 C; c

Analysis of urinary proteins by multidimensional liquid chromatography of ICAT-labeled peptides. Results obtained by the µLC approach demonstrated that there were several qualitative differences between normal and Dents urinary proteomes. However, although the peptide ion intensities obtained by LC-ESI-MS can give an indication of analyte amounts, this parameter by itself cannot be taken as an accurate measure of protein abundance, because factors other than amount can influence ion intensity by ESI-MS. Therefore, we used a novel analytic approach, LC-MS of ICAT-labeled peptides, which allows the relative quantitation of proteins ( 15 ). Table 5 shows the quantitative results obtained with this method, and Fig. 4 illustrates representative ICAT ion pairs. These data confirmed the results obtained by the 2DE and µLC analyses and showed that several plasma proteins were present at similar levels in Dents and normal urine when the analysis was standardized to total protein content ( Table 5 ). However, in agreement with the 2DE data, carrier proteins such as HPX, RBP, VDBP, 2 -GP-I, and several IGFBPs were present at higher levels in Dents urine. Several cytokines were also present at higher levels in Dents, in agreement with the results obtained by µLC. However, results disagreed for transferrin, and no ICAT data could be recorded for TTR, OPN, and PEDF. TTR and OPN do not contain a cysteine residue in their coding sequence and therefore cannot be labeled by ICAT reagents, whereas PEDF only has one cysteine-containing tryptic peptide, which might explain why this peptide was not detected among the complex peptide mixture generated by trypsin digestion of the whole sample. Transferrin appeared to be present at higher levels in Dents urine by ICAT, which was at variance with the 2DE data. This probably reflects a saturation of signal in the silver-stained gels. The detection of uromodulin, kininogen, EGF precursor, and others detected at higher levels in normal urine by ICAT agreed with the gel electrophoresis and/or µLC data ( Table 5 and Fig. 4 ).

Table 5. Relative amounts of polypeptides in the urine of Dents and normal subjects' \" U) p1 H/ Z; @! }; V7 ?( N
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Fig. 4. Representative ion pairs used for the relative quantitation of urinary proteins by multidimensional LC of ICAT-labeled peptides. Dents urinary proteins were labeled with the heavy ICAT reagent, whereas normal urinary proteins were labeled with the light reagent. Fifty micrograms of each labeled sample were then mixed, digested with trypsin, and the resultant peptides were analyzed by LC-ESI-MS/MS. The ratio of the heavy (Dents; right peaks) to light (normal; left peaks) pairs was calculated by dividing the ion signal intensities of the first isotopes. Three examples of peptide ion pairs are shown ( A - C ). The difference between the heavy and the light ICAT reagents is 9 Da. Because the peptide ions shown are triply charged, the difference between the ion pairs is 3 m / z units for albumin and VDBP (these peptides contain 1 labeled cysteine residue) and 6 m / z units for IGFBP-7 (this peptide contains 2 labeled cysteine residues). The figure shows that albumin was present at similar levels in both samples ( A ); conversely, VDBP was 11-fold more abundant in Dents urine ( B ), whereas IGFBP-7 was 33 times more abundant in normal urine ( C ).' m# s+ x6 _- j+ e* {4 a4 c
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DISCUSSION
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Several qualitative differences in the proteome profiles between Dents disease and normal urine were found by three complementary proteomic methods: µLC, 2DE, and multidimensional LC of isotopically labeled proteins (ICAT). 2DE is commonly used to compare protein patterns of related samples, although there are limitations to this technique ( 14 ). For example, protein separation by 2DE is problematic for hydrophobic, very large or small, and very basic or acidic proteins. In addition, not all proteins can be visualized by silver or other staining methods ( 12 ). For example, acidic proteins such as OPN have been found to be poorly stained with silver ( 12 ), which might explain why this protein was identified by the µLC approach but not by 2DE analysis. Nevertheless, 2DE is widely used for protein separation in proteomics and is a well-established method for the visualization of proteomes; the results are easy to interpret, and 2DE provides a simple platform from which a qualitative and quantitative comparison of related proteomes can be made ( 30, 34 ). As an alternative to 2DE, LC can be used before mass spectrometry for the separation of proteins ( 49 ) or their proteolytic products ( 51 ). An advantage of LC-based proteomic approaches is that polypeptide detection by LC-MS is less discriminatory of the physicochemical properties of the analyte and has a higher sensitivity for polypeptides that are not amenable to 2DE. Moreover, analysis is faster and LC can be connected online to mass spectrometry. However, some large proteins may not elute in a defined peak or might precipitate during the chromatographic run, and so they may not be detectable by this method. Moreover, quantitation of LC-separated polypeptides, although feasible, is more challenging and demanding, as proteins have to be either labeled with ICAT ( 15, 49 ) or other isotopically different compounds or purified from cells grown in media containing isotopically labeled amino acids ( 41 ). ICAT reagents label cysteine residues; hence, proteins that do not contain this amino acid cannot be quantified or detected by using this method. In this study, several proteins, including TTR, OPN, PDEF, and several apolipoproteins that were detected by µLC with subsequent LC-ESI-MS/MS, could not be quantified by ICAT. Consequently, due to the inherent physicochemical heterogeneity of proteins, which makes possible their diverse biological functions, a single analytic approach may not be sufficient for a comprehensive analysis of all proteins in a sample. Thus proteomic studies should benefit from using several separation methods before mass spectrometric analysis, as illustrated in the present study
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Defects in the ClC-5 protein abolish megalin-mediated endocytosis of proteins, causing low-molecular-mass proteinuria ( 5, 13, 52 ). Indeed, the quantitatively elevated excretion of specific proteins in the urine of Dents disease patients and animal models of this disease is well documented ( 9, 36, 37, 43, 50 ). However, the present study describes the first detailed examination of the qualitative differences in protein excretion and their relative abundance in Dents vs. normal urine. Because disruption of the ClC-5 channel significantly reduces the rate of megalin-mediated endocytosis ( 5 ), our results may also be relevant to the function of this pathway.
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We found that several plasma proteins were present at similar levels in Dents and normal urine when the analysis was standardized to the total amount of protein present. In contrast, carrier proteins seem to be present at higher levels in the urine of Dents patients compared with normal subjects. In a normal kidney, most proteins present in the glomerular filtrate are reabsorbed in the first segment of the proximal tubule by receptor-mediated endocytosis. However, some proteins fail to be reabsorbed, so that trace amounts of protein are normally present in urine. Our results can be interpreted by assuming that the endocytic apparatus in proximal tubular cells has a greater affinity for carrier proteins than for other classes of proteins. Thus certain carrier proteins, such as HPX, VDBP, RBP, TTR, and 2 -GP I, which transport important bioactive molecules and vitamin precursors in plasma and are of a size that allows them to be freely filtered at the glomerulus, may be almost completely reabsorbed by cells expressing functional ClC-5 channels and are therefore excreted less in normal urine compared with filtered proteins that have a lower affinity for megalin. However, it should be remembered that because we standardized to total protein amount and the total protein concentration in Dents urine is some 30-60 times higher than in normal urine, even proteins that appear to be present in similar amounts with respect to protein concentration are, in fact, being excreted in greater absolute amounts by these patients.; q- {& E1 [& A# t# g+ I$ [) v

We also found several proteins at higher levels in normal urine compared with Dents urine. Many of these proteins are endogenously expressed in the kidney and are secreted. Secreted proteins comprise a significant amount of the total urinary proteome in normal subjects, because tubular cells reabsorb most filtered plasma proteins. In contrast, plasma proteins dominate the proteome of Dents urine, and thus proteins of renal origin represent a smaller proportion of the total Dents urinary proteome compared with normal urine. Consequently, if a protein is detected in normal urine at higher levels than in Dents urine, it can be inferred that this protein is potentially of renal origin and has not been filtered from plasma. For example, we have found that EGF precursor, OPN, kininogen, uromodulin, the poly-Ig receptor, megalin, cubilin, and CD59, among others, are found at higher levels in normal urine; the kidney is known to be a source of several of these proteins. Those such as CD59, the poly-Ig receptor, lithostathine, and IGFBP-7 are so far less well characterized in urine, but our interpretation is that they are primarily of renal origin.: W& l8 q3 N0 `! L, m" s0 w

Proteins that we found in Dents urine at higher levels than in normal urine include cytokines, apolipoproteins, and complement components. Interestingly, these protein classes have all been implicated in progressive renal injury ( 20, 21, 32, 33, 35, 40 ). In contrast, other potentially bioactive peptide precursors, such as kininogen, OPN, and EGF precursor, were found in normal urine at higher levels than in Dents urine when the analysis was standardized to total protein amount, which, in agreement with published data ( 23, 24, 27 ), suggests that the source of these proteins is probably renal. Because receptors for some of these, and other, peptides have been located on the luminal side of tubular epithelial cells ( 16, 25, 44 ), this alteration in the relative concentrations of bioactive peptides in tubular fluid could contribute to a disturbance of normal cell signaling mechanisms. In addition, regulatory proteins such as CD59 (which prevents complement complex formation) ( 48 ), uromodulin (which binds and perhaps modulates cytokine activity) ( 18 ), lithostathine (which binds inorganic crystals) ( 10 ), IGFBP-7 (which binds IGF) ( 39 ), and latent TGF- -binding protein (which binds latent TGF- ) ( 22 ) are present at lower levels in Dents urine relative to total protein compared with normal urine. Thus the proposed regulatory role of these proteins could be overwhelmed in Dents patients and such changes in urine composition, in part reflecting tubular fluid, might contribute to the commonly observed progression of renal failure in this and other forms of renal Fanconi syndrome.

In conclusion, our results indicate that the cubilin-megalin-mediated endocytic pathway has a greater affinity for carrier proteins and potentially toxic proteins of plasma origin than for other classes of proteins. In addition, these observations suggest that the salvage of vitamins and prosthetic groups may be of particular physiological importance and that another important role of the receptor-mediated endocytic pathway is to protect more distal portions of the nephron from the potentially harmful effects of some filtered plasma polypeptides. It is therefore likely that the cubilin-megalin receptor system has different affinities for different proteins. Alternatively, the apparent specificity might be explained by the existence of yet other, and so far unidentified, receptors that participate in protein endocytosis along the proximal tubule.2 G6 m1 e1 S# S4 m1 B0 |0 ]0 n
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GRANTS

This work was supported by the Ludwig Institute for Cancer Research, St. Peters Trust for Bladder and Kidney Research (P. R. Cutillas and R. J. Unwin), the Sir Jules Thorn Charitable Trust (A. G. W. Norden), and Division of Research Resources Grant RR-01614.

Table 2. Proteins found at similar levels in Dents and normal urine by 2DE

ACKNOWLEDGMENTS- q1 x: |5 ]- ]9 I  R

We are grateful to Drs. T. Feest, S. Rigden, and R. Walters for access to the patients under their care, and we also thank Dr. John Timms for critical comments on the manuscript.
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