J Am Soc Nephrol 11: 632–639, 2000 Characterization and Localization of the Neonatal Fc in Adult Human Kidney

JEAN-PHILIPPE HAYMANN,* JEAN-PIERRE LEVRAUD,† SANDRINE BOUET,* VINCENT KAPPES,* JACQUELINE HAGEGE,*` GENEVIEVE` NGUYEN,* YICHUN XU,* ERIC RONDEAU,* and JEAN-DANIEL SRAER* *Service de Ne´phrologie A, Assistance Publique-Hoˆpitaux de Paris, Institut National de la Sante´etdela Recherche Me´dicale U489 et Association Claude Bernard, Hoˆpital Tenon, and † Institut National de la Sante´ et de la Recherche Me´dicale U277, Institut Pasteur, Paris, France.

Abstract. The binding of Fc fragments of Ig on glomerular by flow cytometry, reverse transcription-PCR, Western blot- epithelial cells (GEC) was observed previously, but the recep- ting, and by the pH dependence of the binding of heat-aggre- tor could not be identified. In immunofluorescence and immu- gated IgG. Because it is well established that the FcRn is nohistochemical studies using normal adult human kidney sec- involved in IgG transcytosis, it is hypothesized that the FcRn in tions, the presence of the so-called neonatal (FcRn) the kidney may play a role in the reabsorption of IgG. Ongoing was demonstrated on GEC as well as in the brush border of studies should clarify the role of the FcRn as a potential target proximal tubular cells. FcRn transcripts were also detected on for immune complexes on GEC and should assess its relevance isolated glomeruli by reverse transcription-PCR. Using an im- in physiology and pathology. mortalized GEC line, the presence of the FcRn was confirmed

Immune complex deposition is encountered in many glomer- transcribed in many adult tissues (7,8,19), has been identified ulonephritides. In membranous nephropathy, those deposits as the “IgG protection” receptor hypothesized by Brambell et occur between the glomerular basement membrane and vis- al. (20) to explain the paradoxically long half-life of IgG, ceral glomerular epithelial cells (GEC). This observation has relative to the half-lives of other plasma . This has led led investigators to search for a specific IgG receptor on the to the hypothesis that FcRn is expressed on endothelial cells surface of GEC. It was reported that soluble aggregated IgG (21–23). To date, the expression of FcRn on extracellular Ј (AgIgG) or Fc fragments, but not F(ab )2 fragments, could bind plasma membranes has been reported only for enterocytes and to GEC in culture (1) and in tissue sections (2). However, no hepatocytes, whereas FcRn appears to be localized exclusively receptors responsible for this binding were identified, and in the cytoplasm, associated with acidified endosomes, in syn- subsequent searches for the known Fc␥ receptors CD16, CD32, cytiotrophoblast cells and some endothelial cells (13,23). and CD64 on GEC yielded negative results (3–6). FcRn mRNA has been detected by Northern blot analysis of Another Fc␥ receptor, known as the neonatal Fc receptor human kidneys (8), but the precise localization of this receptor (FcRn), has been cloned from rodents (7) and human subjects on the different structures of the kidney has not been investi- (8). This receptor is an MHC class I-like membrane gated. Therefore, we raised the question of whether the binding ␤ associated with 2-microglobulin. FcRn is considered to be of AgIgG or Fc fragments to GEC may be linked to the involved in IgG transport from the blood of the mother to that presence of this receptor. We show here that this receptor is of the fetus during pregnancy (8–13) and from the milk of the expressed on GEC in vivo and in vitro, as well as on the brush mother to the neonate during lactation (14–16). The ability of border of proximal tubular cells. These data raise interesting this receptor to bind IgG with higher affinity at the acidic pH questions regarding the relevance of this receptor in physio- encountered in the gut lumen, compared with the neutral logic processes and in some glomerular diseases, such as plasma pH, is thought to be important in the latter function membranous nephropathy. (16–18). However, FcRn function is not restricted to the trans- fer of IgG from mother to offspring. Indeed, FcRn, which is Materials and Methods Reagents Received February 10, 1998. Accepted September 1, 1999. The rabbit anti-FcRn was a polyclonal antiserum specific Correspondence to Dr. Jean-Philippe Haymann, Service de Ne´phrologie A, for the rat FcRn heavy chain (kindly provided by Dr. Pamela Bjork- Hoˆpital Tenon, 4 Rue de la Chine, 75020 Paris, France. Phone: ϩ3315601 man, California Institute of Technology, Pasadena, CA) (18). Recog- 65 10; Fax: ϩ33 1 56 01 79 68; E-mail: [email protected] nition of the human FcRn by anti-rat FcRn was reported paris.fr previously (13). Normal rabbit serum was used as the negative con- 1046-6673/1104-0632 trol. The soluble FcRn was described previously (24) and was also Journal of the American Society of Nephrology kindly provided by Dr. Pamela Bjorkman. AgIgG was obtained by Copyright © 2000 by the American Society of Nephrology heating at 63°C for 30 min, as described (1). Anti-CD16, -CD32, and J Am Soc Nephrol 11: 632–639, 2000 FcRn Expression in Human Kidney 633

Ј -CD64 antibodies, goat anti-human F(ab )2, and human Fc fragments volume. Forty rounds of amplification, each consisting of 30 s at were purchased from Jackson ImmunoResearch (West Grove, PA). 94°C, 30 s at 60°C, and 30 s at 72°C, were then performed in a 9600 Ј FITC-conjugated anti-rabbit IgG, anti-human F(ab )2, and anti-mouse GeneAmp thermocycler (Perkin Elmer, Foster City, CA). IgG were obtained from Amersham (Orsay, France). The following oligonucleotides (purchased from Eurogentec) were used, yielding an expected 369-bp product from cDNA: FcRn1, Cell Culture 5Ј-CAAAGCTTTGGGGGGAAAAG-3Ј (hybridizing in the ␣1 do- Ј Ј Human GEC were isolated from normal tissue obtained from main); FcRn2, 5 -TGCAGGTAAGCACGGAAAAG-3 (hybridizing ␣ nephrectomies and were characterized as described previously (25). in the 3 domain). Sequencing of the PCR product was performed The cells were cultured in RMPI 1640 (Life Technologies) containing with the ABI Prism dye terminator reaction kit (Perkin Elmer), using 10% heat-inactivated fetal calf serum and 2 mM L-glutamine, and they the recommended protocol; the product was analyzed using a 373A were used between passages 3 and 4. A stable GEC cell line, E56 automated DNA sequencer (Applied Biosystems, Foster City, CA). 10A1 (hereafter referred to as E56), with a phenotype similar to that of primary cultures of GEC and in vivo (25,26) was used Immunoblot Analysis between passages 60 and 80. A human choriocarcinoma cell line Cell membranes from E56 cells and isolated glomeruli (obtained (BEWO) was obtained from the European Collection of Animal Cell after sieving) were prepared as described previously (29). Briefly, the Cultures (no. 86082803) at passage 196 and was cultured in Ham’s cells were rapidly washed three times with cold Krebs-Henseleit

F-12 medium (Life Technologies) containing 2 mM glutamine and buffer (118 mM NaCl, 5 mM KCl, 1.1 mM MgSO4, 2.5 mM CaCl2, 10% fetal calf serum. 1.2 mM KH2PO4, 25 mM NaHCO3, pH 7.4) and scraped into homog- enization buffer (5 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose, Immunofluorescence Study 500 U/ml Trasylol (Bayer Pharma, Puteaux, France), 1 mM ethylene Normal portions of noninvolved poles from three tumor nephrec- glycol-bis(␤-aminoethyl ether)-N,N,NЈ,NЈ-tetra-acetic acid, and 1 mM tomy specimens were studied. The tissues were rapidly frozen in phenylmethylsulfonyl fluoride). The cells were homogenized at 0°C liquid nitrogen, and 2-␮m-thick cryostat sections were fixed in 4% in a Teflon Potter homogenizer. Two milliliters of the homogenate paraformaldehyde for 10 min and washed in phosphate-buffered sa- were loaded on 1 ml of 20 mM Tris-HCl, pH 7.5, containing 1.45 M line (PBS). The sections were incubated with the rabbit antiserum to sucrose. After centrifugation at 35,000 ϫ g for 30 min, the membranes FcRn (at a dilution of 1:40) for 30 min at room temperature, washed at the interface were collected, pelleted at 40,000 ϫ g for 20 min, and extensively with PBS, and incubated with FITC-anti-rabbit IgG for 30 washed in 10 mM Hepes, pH 7.5, containing 0.2 mM CaCl2,5mM min. Double staining was performed using a monoclonal antibody to MgCl2, 250 U/ml Trasylol, and 0.5 mM phenylmethylsulfonyl fluo- CD31 (dilution 1:100; Dako, Glostrup, Denmark), an endothelial cell ride. The cell membranes were extracted in 5% sodium dodecyl marker, and a Texas red-labeled anti-mouse IgG (Vector Laboratories, sulfate. Protein concentrations in the extracts were determined by the Burlingame, CA) for detection. The slides were then washed and method of Peterson (30). The extracts and recombinant rat FcRn were photographs were taken using immunofluorescence microscopy. resolved on 12% polyacrylamide denaturing gels and transferred to nylon membranes. The membranes were blocked with 5% nonfat milk Immunohistochemical Study in PBS and probed with rabbit antiserum to FcRn (dilution 1:500) or nonimmune control serum (dilution 1:500) overnight at 4°C. Unbound The rabbit polyclonal anti-FcRn was detected using the biotin- antibodies were removed by washing in PBS with 0.05% (vol/vol) avidin-peroxidase-coupled technique. In brief, the tissue sections were Tween 20. A second antibody, alkaline phosphatase-conjugated goat blocked with 10% normal human serum before incubation with the anti-rabbit IgG, was then applied for 30 min at 37°C, and the unbound specific rabbit polyclonal anti-FcRn antibody (dilution 1:320) for 1 h antibody was removed by washing as described above. Immunoreac- at room temperature. After being washed with PBS, the sections were tivities were revealed with nitroblue tetrazolium/5-bromo-4-chloro-3- incubated with a biotinylated anti-rabbit antibody (Dakopatts, indolyl phosphate substrate (Promega). Glostrup, Denmark) and then incubated with avidin coupled to per- oxidase (Amersham, Buckinghamshire, United Kingdom), which was Flow Cytometric Analysis detected with 3-amino-9-ethylcarbazole in the presence of H2O2. Sections were then counterstained with hematoxylin. Negative control E56 cells were detached with 5 mM ethylenediaminetetra-acetic samples were prepared using nonimmune rabbit antiserum (dilution acid, washed three times with PBS, pH 7.4, and incubated for 30 min 1:320). with either rabbit anti-FcRn, nonimmune rabbit serum (as a negative control), anti-CD16, anti-CD32, or anti-CD64. After washing with Reverse Transcription-PCR PBS, the cells were incubated with FITC-conjugated goat anti-rabbit Explanted isolated glomeruli from three different specimens were IgG or anti-mouse IgG for1hatroom temperature, washed, and obtained by microdissection, as described (27). Total RNA was ex- analyzed using a flow cytometer (Beckton Dickinson, Mountain tracted from microdissected glomeruli and cultured cells by ultracen- View, CA). Data were analyzed with CellQuest software (Beckton trifugation on a CsCl cushion (28). cDNA was synthesized from 10 Dickinson). Dead cells were excluded on the basis of propidium ␮ iodide incorporation. g of total RNA, using 100 pmol of (dT)17 primer, 25 U of RNasin (Promega, Madison, WI), and 10 U of avian myeloblastosis virus reverse transcriptase (Boehringer, Mannheim, Germany), in the buffer Labeling of AgIgG and Binding Assays provided. For control samples, reverse transcriptase was omitted. AgIgG was labeled with Na125I, by the Iodogen method (Pierce, cDNA was diluted with water to a final volume of 100 ␮l. PCR was Rockford, IL), to a specific activity of 0.5 Ci/␮mol. Primary GEC performed using the following mixture: 25 U/ml Goldstar Taq DNA cultures or E56 cells were grown to confluence in 100-mm-diameter polymerase (Eurogentec, Seraing, Belgium) in the buffer provided, culture plates (Nunc, Roskilde, Denmark). The cells were detached as ␮ 2.5 mM MgCl2, 0.2 mM dNTP, and 0.5 M levels of each primer. described previously and resuspended in binding buffer (Hanks’ bal- Three microliters of cDNA were used as the template, in a 30-␮l final anced salt solution, with 10 mM Hepes, pH 6.0 or 8.0, containing 634 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 632–639, 2000

Figure 1. Localization of the neonatal Fc receptor (FcRn) in a normal adult kidney section by immunofluorescence and immunohistochemical analyses. A normal adult human kidney section was stained. (A) Staining with the FcRn-specific antiserum was detected using FITC-labeled anti-rabbit IgG antibodies. This labeling indicated a origin in the glomerulus. The brush borders of proximal tubular cells were also labeled. The negative control sample was almost totally dark and therefore is not shown. (B) No labeling was observed when soluble rat J Am Soc Nephrol 11: 632–639, 2000 FcRn Expression in Human Kidney 635

0.25% bovine ). The cells were pelleted, washed, and resuspended in binding buffer at approximately 106 cells/ml. For binding assays, 3 ϫ 105 cells in 400 ␮l were mixed with 125I-AgIgG (106 cpm), with or without unlabeled AgIgG (to measure nonspecific binding). The cells were allowed to bind AgIgG at 4°C for 24 h with gentle stirring, transferred to Eppendorf tubes, and pelleted at 2000 rpm for 2 min at 4°C. After three washes with Hanks’ balanced salt solution, pH 6.0 or pH 8.0, cell-associated radioactivity was counted in a gamma counter. All experiments were performed in triplicate.

Results Distribution of the FcRn in Kidney Tissue Sections We investigated whether, under physiologic conditions, FcRn was expressed by GEC in adult kidneys. First, normal human kidney tissues were stained with an antiserum to rat FcRn in immunofluorescence assays (Figure 1A). The assays Figure 2. Expression of FcRn mRNA by normal adult human glo- demonstrated positive staining in the glomeruli, strongly indi- meruli. Reverse transcription (RT)-PCR, using specific primers for cating GEC distribution, with no staining of parietal epithelial FcRn, was performed on microdissected glomerular RNA extracts cells. Unexpectedly strong staining was also observed on prox- from three different specimens (lanes 2, 3, and 4), and products were imal tubular cells, associated with the brush border. The spec- analyzed on an ethidium bromide-stained agarose gel. Lane 1, PCR ificity of this staining was confirmed by complete inhibition of negative control (water as template); lane 0, 100-bp ladder. A PCR the staining in the presence of soluble FcRn (Figure 1B). No product of the expected size (359 bp) can be clearly observed. staining was detected in the interstitium, in the medulla, or in distal tubular cells. Although the immunofluorescence patterns exclude staining Characterization of the FcRn on Immortalized GEC of mesangial cells, the presence of FcRn on podocytes or To perform functional assays on this receptor, we took endothelial cells is an important matter of debate, because advantage of the generation of an immortalized human GEC endothelial cells in liver and muscles were shown to express line, E56, in our laboratory (25). Before this model could be this receptor (23). To carefully address this question, double considered valid, however, we needed to confirm that the staining with an -specific marker (CD31) and im- expression of Fc receptors mirrored the in vivo situation. munohistochemical assays were performed. Figure 1C indi- Therefore, after having confirmed the binding of AgIgG to E56 cates the different distributions of FcRn and CD31 in glomer- cells in flow cytometric assays (data not shown), we analyzed uli, confirming the podocyte localization of FcRn. The absence the expression of the Fc receptor candidates, namely CD16, of endothelial cell staining was clearly demonstrated in the CD32, CD64, and FcRn. immunohistochemical analyses (Figure 1E), although very First, we investigated the expression of CD16, CD32, CD64, small amounts of FcRn might not have been detected. and FcRn on E56 cells by flow cytometry. As shown in Figure Total microdissected glomerular RNA extracts from three 4, the rabbit antiserum to rat FcRn yielded modest but signif- different specimens were tested for the presence of FcRn icant staining of E56 cells, whereas normal rabbit serum did mRNA. Reverse transcription (RT)-PCR was performed using not. In contrast, monoclonal antibodies to the three myeloid primers specific for human FcRn. As shown in Figure 2, a PCR Fc␥ receptors (CD16, CD32, and CD64) did not stain E56 cells product of the expected size was obtained from all lines. at all. Sequencing of this PCR fragment demonstrated 100% identity Total RNA was extracted from either E56 cells, primary with the previously reported sequence of human FcRn (8). To cultured GEC, or a trophoblastic cell line chosen as a positive biochemically assess the specificity of the FcRn antiserum, we control (BEWO). As shown in Figure 5, a RT-PCR product of performed Western blotting of isolated glomerular extracts, the expected size was obtained from all lines. There was no which revealed the presence of two bands of approximately 45 genomic DNA contamination, because PCR performed on kD, consistent with two glycosylated forms of the FcRn heavy GEC total RNA without reverse transcriptase yielded negative chain (Figure 3, lane 1). results. Furthermore, the two PCR primers used are expected to

FcRn was first incubated with the FcRn-specific antiserum. (C) Double-fluorescence staining using an anti-CD31 antibody (red) and the FcRn antiserum (green) indicated different localizations in the glomerulus. (E) Detection of the FcRn-specific antiserum by a biotin-avidin- peroxidase-coupled technique indicated staining of the podocytes at the periphery of the glomerulus (arrows). On the other side of the glomerular basement membrane, endothelial cells in capillaries (labeled C) were not stained. Proximal tubular cells (TCP) were labeled. No staining of parietal glomerular epithelial cells (GEC) (arrowhead) was demonstrated. (D) The negative control is shown. Magnification: ϫ250 in A through D; ϫ600 in E. 636 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 632–639, 2000

Figure 4. Detection of FcRn on the surface of immortalized GEC by Figure 3. Detection of FcRn in membrane extracts of immortalized flow cytometry. (Top Left) E56 cells were stained with a rat FcRn- GEC or isolated glomerular extracts by Western blotting under non- specific polyclonal antiserum (solid line) or with normal rabbit serum reducing conditions. Protein membrane extracts (40 ␮g/lane) of E56 (dashed line), which was detected with FITC-labeled anti-rabbit IgG cells (lanes 3 and 4), glomerular extracts (40 ␮g/lane) (lanes 1 and 2), antibodies. The other three panels show E56 cells that were stained or soluble truncated rat FcRn (0.01 ␮g/lane) (lanes 5 and 6) were with monoclonal antibodies specific for either CD16, CD32, or CD64, probed with FcRn-specific polyclonal antiserum (lanes 1, 3, and 5) or as indicated, with detection using FITC-labeled anti-mouse IgG anti- with control rabbit serum (lanes 2, 4, and 6), using a working dilution bodies (solid lines). The dashed lines represent fluorescence with the of 1:500. The positions of molecular mass markers are indicated on secondary antibodies alone. the left. The data clearly indicate a 40- to 45-kD band with several glycosylated products for the recombinant rat FcRn, with the 80-kD band being an FcRn dimer (lane 5). There are probably at least two glycosylated forms for human FcRn in glomerular extracts (lane 1) under the latter conditions (Figure 6). Similar results were and E56 cell extracts. The 60-kD band is currently unidentified. The obtained using primary cultured GEC (data not shown). control serum did not react with any band. To further demonstrate the identity of the AgIgG binding site with the FcRn, we performed an inhibition assay with the polyclonal antiserum to rat FcRn. The binding of 125I-AgIgG to GEC at pH 6 was inhibited by 80% in the presence of this hybridize with two different exons. No RT-PCR product for specific antiserum, compared with nonimmune serum (Figure CD16, CD32, or CD64 could be detected in GEC using spe- 7). cific primers (data not shown). Moreover, we performed an inhibition experiment at pH 6 Finally, Western blotting experiments on E56 cell mem- using increasing concentrations of unlabeled AgIgG, which brane extracts under nonreducing conditions revealed the pres- further demonstrated that the binding of 125I-AgIgG to GEC ence of a wide specific band of 40 to 45 kD (Figure 3, lane 3), was specific and saturable. Because AgIgG is heterogeneous in which may represent different glycosylated forms of the FcRn size (31), it is difficult to perform Scatchard analysis of such heavy chain, similar to the recombinant rat FcRn (Figure 3, data. Postulating a molecular mass of AgIgG of approximately 6 Ϫ6 Ϫ7 lane 5). As shown for recombinant rat FcRn, the 80-kD band 10 D, we estimated the Kd to be approximately 10 to 10 indicates an FcRn dimer form (Figure 3, lane 5). Taken to- M, with 2500 binding sites/cell (data not shown). gether, these data demonstrate that the E56 cell line expresses the FcRn but not the other Fc␥ receptors (CD16, CD32, and Discussion CD64), similar to GEC expression in vivo. Our study localizes, for the first time, the FcRn in normal A characteristic feature of the FcRn is its higher affinity for adult human kidneys on podocytes as well as on proximal Fc at pH 6.0 to 6.5, compared with pH 7.5 to 8.0 (8,12,17,19), tubular cells. The presence of this receptor was indicated by a property that is thought to be responsible for the trafficking immunofluorescence staining and immunohistochemical anal- of IgG from the gut lumen to the bloodstream in neonates. ysis of human kidney biopsy samples, Western blotting, and Using 125I-AgIgG, we investigated the pH dependence of the RT-PCR assays of microdissected glomeruli. The expression binding of AgIgG on E56 cells. The results showed that al- of the FcRn on a GEC cell line was also demonstrated (by flow though 125I-AgIgG could bind to GEC in a specific manner at cytometry, PCR, and Western blotting), and was functional, both pH 6 and pH 8, specific binding levels were much lower since pH-dependent binding of AgIgG was demonstrated. J Am Soc Nephrol 11: 632–639, 2000 FcRn Expression in Human Kidney 637

Figure 5. GEC expression of FcRn mRNA. Total RNA from the Figure 7. Blockade of the binding of AgIgG on E56 cells by FcRn- human trophoblast cell line BEWO (lane 1), E56 cells (lane 2), and specific antiserum. E56 cells were first incubated either with the GEC in primary culture (lane 3) were subjected to RT-PCR using polyclonal FcRn-specific antiserum (immune) or with normal rabbit specific FcRn primers and were analyzed on an ethidium bromide- serum (control). After 30 min, 125I-AgIgG was added and binding was stained agarose gel. Lane 4, PCR negative control (water as template); measured as for Figure 6. Values are the mean and SD of triplicate lanes 5 and 6, controls for genomic DNA contamination (PCR per- experiments. formed on RNA without reverse transcriptase); lanes 0 and 7, 100-bp ladder. CD16 and CD32 (two other low-affinity Fc receptors) bind in vitro only IgG aggregates in solution and no IgG monomers. This finding is fully consistent with our own observations and previous reports, in which only AgIgG (1) or IgG-coated polystyrene latex particles (2) bound to podocytes ex vivo. This finding also provides an explanation for why no human IgG deposits (on podocytes) are detected in normal glomeruli in vivo; IgG must first cross the glomerular basement membrane and then at least dimerize. However, after immune complexes are immobilized in the extracellular matrix, FcRn dimerization, which enhances IgG binding affinity (32–34), may be facili- tated. FcRn is considered to play a key role in IgG transcytosis in many organs. In podocytes, a cellular mechanism of transcy- tosis has already been reported for the C5b-9 membrane attack complex (35). Because endocytosis occurs in clathrin-coated areas after incubation of AgIgG with GEC, as observed in vitro by electron microscopy (1), FcRn-mediated IgG transcytosis in GEC may be proposed. Therefore, this receptor might be involved in the clearance of immune complexes present in Figure 6. pH-dependent binding of aggregated IgG (AgIgG) by E56 cells. E56 cells were incubated for 24 h at 4°C with iodinated AgIgG, pathologic conditions, such as membranous nephropathy. at pH 6.0 or 8.0. Assays were performed in the absence (Ϫ)or Another function might be attributed to this receptor in the presence (ϩ) of a 1000-fold excess of unlabeled IgG, to assess the kidney. Proteins that are filtered through the glomeruli, such as specificity of the binding. Cell-associated radioactivity was then mea- albumin, are reabsorbed primarily in renal proximal tubular sured in a gamma counter after three washes. Values are the mean and cells, where they are catabolized (36). The presence of the SD of triplicate experiments. FcRn in the brush border suggests reabsorption of IgG or Fc fragments. Indeed, it was demonstrated that infused Fc frag- ments were reabsorbed in renal proximal tubular cells in rats The absence of IgG background staining on podocytes in (37). However, the Fc catabolic degradation process seemed immunocytochemical assays, despite the presence of an Fc not to be located in normal kidneys, inasmuch as the serum receptor on those cells, is explained by the low affinity of the half-life of Fc fragments was not altered by nephrectomy (38). receptor. Indeed, this low-affinity receptor (especially at pH This absence of Fc fragment degradation suggests a recycling 7.2 to 7.4) binds only multimers of IgG, in the same way that process for IgG or Fc fragments at this location mediated by 638 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 632–639, 2000

FcRn. This would agree well with the recently established role 7. Simister NE, Mostov KE: An Fc receptor structurally related to of FcRn in the protection of plasma IgG from catabolism MHC class I antigens. Nature 337: 184–187, 1989 (21–23). Indeed, IgG exhibits a long survival time, relative to 8. Story MC, Mikulska JE, Simister NE: A major histocompatibil- ␤ ity complex class I-like Fc receptor cloned from human placenta: other plasma proteins (20); however, in 2-microglobulin knockout mice, in which the FcRn is not functional, IgG is Possible role in transfer of from mother to cleared at the same accelerated rate as albumin (21). It can fetus. J Exp Med 180: 2377–2381, 1994 therefore be hypothesized that some IgG crosses the glomeru- 9. Roberts MR, Guenthert M, Rodewald RJ: Isolation and charac- lar basement membrane under physiologic conditions and that terization of the Fc receptor from the fetal yolk sac of the rat. J Cell Biol 111: 1867–1876, 1990 FcRn on apical proximal tubular cells allows endocytosis and 10. Ahouse JJ, Hagerman CL, Mittal P, Gilbert DJ, Copeland NG, transport of intact IgG back to the circulation, with the kidney Jenkins NA, Simister NE: Mouse MHC class I-like Fc receptor playing a role in this protection of IgG from catabolism. Such encoded outside the MHC. J Immunol 151: 6076–6088, 1993 a hypothesis is contrary to the widely held view that the 11. Leach JL, Sedmak DD, Osborne JM, Rahill B, Lairmore MD, glomerular filter is an absolute barrier for IgG, a concept that Anderson CL: Isolation from human placenta of the IgG trans- is supported by the absence of detectable IgG in the ultrafiltrate porter, FcRn, and localization to the syncytiotrophoblast. J Im- (37). However, given the large volume of plasma filtered munol 157: 3317–3322, 1996 through the kidneys, even a minute leakage of IgG through the 12. Simister NE, Story CM, Chen HL, Hunt JS: An IgG-transporting barrier might result in significant daily loss from the total IgG Fc receptor expressed in the syncytiotrophoblast of human pla- pool if those molecules were not recycled, with IgG remaining centa. Eur J Immunol 26: 1527–1531, 1996 undetectable in the ultrafiltrate by conventional methods. A 13. Kristoffersen EK, Matre R: Co-localization of the neonatal Fc␥ ␤ testable prediction of this hypothesis would be that in 2- receptor and IgG in human placenta term syncytiotrophoblasts. microglobulin knockout mice, the kidney would be an impor- Eur J Immunol 26: 1668–1671, 1996 tant site for IgG catabolism. Additional work is required to 14. Rodewald R: Intestinal transport of antibodies in the newborn rat. determine whether this receptor has functional relevance under J Cell Biol 58: 189–211, 1973 physiologic conditions and/or in some human glomerulone- 15. Simister NE, Rees AR: Isolation and characterization of an Fc phritides, in which subepithelial IgG deposits are found in the receptor from neonatal rat small intestine. Eur J Immunol 15: glomeruli and IgG is observed in the urine. 733–738, 1985 16. Jakoi ER, Cambier J, Saslow S: Transepithelial transport of maternal antibody: Purification of IgG receptor from newborn rat Acknowledgments intestine. J Immunol 135: 3360–3364, 1985 This work was supported by the Delegation de la Recherche 17. Rodewald R: pH-dependent binding of immunoglobulins to in- Clinique Assistance Publique-Hoˆpitaux de Paris (Grant CRC97187). testinal cells of the neonatal rat. J Cell Biol 99: 159–164, 1976 Dr. Levraud is the recipient of a fellowship from the Pasteur Institute. 18. Raghavan M, Chen MY, Gastinel LN, Bjorkman PJ: Investiga- We are very grateful to Dr. Pamela Bjorkman for precious reagents. tion of the interaction between the class I MHC-related Fc We thank Drs. Marie-Claire Gubler, Gabriel Gachelin, Pierre Ver- receptor and its immunoglobulin G ligand. 1: 303–315, roust, Jean Kanellopoulos, and Philippe Kourilsky for helpful com- 1994 ments and critical reading. We thank Madeleine Delauche, Francoise 19. Blumberg RS, Koss T, Story CM, Barisani D, Polischuk J, Lipin Delarue, and Latifa Bouzhir for expert technical assistance. A, Pablo L, Green R, Simister NE: A major histocompatibility complex class I-related Fc receptor for IgG on rat hepatocytes. References J Clin Invest 95: 2397–2402, 1995 1. Mancilla-Jimenez R, Appay M, Bellon B, Kuhn J, Bariety J, 20. Brambell FWR, Hemmings A, Morris IG: A theoretical model of Druet P: IgG Fc membrane receptor on normal human glomer- gamma globulin catabolism. Nature 203: 1352–1355, 1964 ular visceral epithelial cells. Virchows Arch A Pathol Anat His- 21. Junghans RP, Anderson CL: The protection receptor for IgG topathol 404: 139–158, 1984 catabolism is the ␤ -microglobulin-containing neonatal intestinal 2. Mizoguchi Y, Horiuchi Y: Localization of IgG-Fc receptors in 2 transport receptor. Proc Natl Acad Sci USA 93: 5512–5516, 1996 human renal glomeruli. Clin Immunol Immunopathol 24: 320– 22. Ghetie V, Hubbard JG, Kim JK, Tsen MF, Lee Y, Ward ES: 329, 1982 Abnormally short serum half-lives of IgG in ␤ -microglobulin 3. Aarli A, Matre R, Thunold S: IgG Fc receptors on epithelial cells 2 deficient mice. Eur J Immunol 26: 690–696, 1996 of distal tubuli and on endothelial cells in human kidney. Int Arch 23. Borvak J, Richardson J, Medesan C, Antohe F, Radu C, Simio- Allergy Appl Immunol 95: 64–69, 1991 4. Sedmak DD, Davis DH, Singh U, van de Winkel JG, Anderson nescu M, Ghetie V, Ward ES: Functional expression of the MHC CL: Expression of IgG Fc receptor antigens in placenta and on class I-related receptor, FcRn, in endothelial cells of mice. Int endothelial cells in humans. Am J Pathol 138: 175–181, 1991 Immunol 10: 1289–1298, 1998 5. Tuijnman WB, Wichen DFV, Schuurman HJ: Tissue distribution 24. Gastinel LN, Simister NE, Bjorkman PJ: Expression and crys- of human IgG Fc receptors CD16, CD32 and CD64: An immu- tallization of a soluble and functional form of an Fc receptor nohistochemical study. Acta Pathol Microbiol Immunol Scand related to class I histocompatibility molecules. Proc Natl Acad 101: 319–329, 1993 Sci USA 89: 638–642, 1992 6. Morcos M, Ha¨nsch GM, Scho¨nermark M, Ellwanger S, Ha¨rle M, 25. Delarue F, Virone J, Hage`ge J, Lacave R, Peraldi MN, Adida C, Heckl-O¨ streicher B: Human glomerular mesangial cells express Rondeau E, Feunteun J, Sraer JD: Stable cell line of T-SV40 CD16 and may be stimulated via this receptor. Kidney Int 46: immortalized human glomerular visceral epithelial cells. Kidney 1627–1634, 1994 Int 40: 906–912, 1991 J Am Soc Nephrol 11: 632–639, 2000 FcRn Expression in Human Kidney 639

26. Krishnamurti U, Chen Y, Michael A, Kim Y, Fan WW, Wies- murine intestinal Fc receptor. Eur J Immunol 24: 2429–2434, lander J, Brunmark C, Rondeau E, Sraer JD, Delarue F, Tsilibary 1994 EC: Integrin-mediated interactions between primary/T-SV40 im- 33. Raghavan M, Wang Y, Bjorkman P: Effect of receptor dimer- mortalized human glomerular epithelial cells and type IV colla- ization on the interaction between the class I major histocompat- gen. Lab Invest 74: 650–657, 1996 ibility complex-related Fc receptor and IgG. Proc Natl Acad Sci 27. Peten EP, Striker LJ, Larome A, Elliott SJ, Yang CW, Striker USA 92: 11200–11204, 1995 GE: The contribution of increased collagen synthesis to glomer- 34. Burmeister WP, Gastinel LN, Simister NE, Blum ML, Bjorkman ␣ ulosclerosis: A quantitative analysis of 2IV collagen mRNA PJ: Crystal structure at 2.2 Å resolution of the MHC-related expression by competitive polymerase chain reaction. J Exp Med neonatal Fc receptor. Nature 372: 336–343, 1994 176: 1571–1576, 1992 35. Kerjaschki D, Schulze M, Binder S, Kain R, Ojha PP, Susani M, 28. Chirgwin JM, Pryzbyla AE, MacDonald KK, Rutter WJ: Isola- Horvat R, Baker PJ, Couser WG: Transcellular transport and tion of biologically active ribonucleic acid from sources enriched membrane insertion of the C5b-9 membrane attack complex of in ribonucleases. Biochemistry 18: 5294–5299, 1979 complement by glomerular epithelial cells in experimental mem- 29. Nguyen G, Self SJ, Camani C, Kruithof EKO: Demonstration of branous nephropathy. J Immunol 143: 546–552, 1989 a specific clearance receptor for tissue-type plasminogen activa- tor on rat Novikoff hepatoma cells. J Biol Chem 267: 6249– 36. Park CH, Maack T: Albumin absorption and catabolism by 6256, 1992 isolated perfused proximal convoluted tubules of the rabbit. 30. Peterson GL: A simplification of the protein assay method of J Clin Invest 73: 767–777, 1984 Lowry et al. which is more generally applicable. Anal Biochem 37. Druet P, Bariety J, Laliberte F, Bellon B, Belair M-F, Paing M: 83: 346–356, 1977 Distribution of heterologous antiperoxidase antibodies and their 31. Knutson DW, Kijlstra A, Es LAV: Association and dissociation fragment in the superficial renal cortex of normal Wistar-Munich of aggregated IgG from rat peritoneal macrophages. J Exp Med rats. Lab Invest 39: 623–631, 1978 145: 1368–1381, 1977 38. Arend WP, Silverblatt FJ: Serum disappearance and catabolism 32. Kim JK, Tsen M-F, Ghetie V, Ward ES: Localization of the site of homologous immunoglobulin fragments in rats. Clin Exp of the murine IgG1 molecule that is involved in binding to the Immunol 22: 502–513, 1975