FcRn-mediated transcytosis of immunoglobulin G in human renal proximal tubular epithelial cells

Noriyoshi Kobayashi1,2, Yusuke Suzuki1,2, Toshinao Tsuge1,2, Ko Okumura2, Chisei Ra2, and Yasuhiko Tomino1

1 Division of Nephrology, Department of Internal Medicine, and 2 Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the kidney, proteins filtered through glomeruli are reabsorbed by endocytosis along the proximal tubules to avoid renal loss of large amounts of proteins. Recently, neonatal Fc receptor (FcRn), which is involved in the transport of IgG across several epithelial and endothelial cells, was reported to be expressed in renal proximal tubular epithelial cells (RPTECs). However, there has been no direct evidence for receptor-mediated endocytosis of IgG in human RPTECs. To explore physiological roles of FcRn in the proximal tubules, we used the human RPTECs to examine IgG transport. FcRn was expressed in RPTECs and physically associated with beta 2-microglobulin, preserving the capacity of specific pH-dependent IgG binding. Human IgG was bound to the cell surface of RPTECs in a pH-dependent manner. The human IgG transport assay revealed that receptor-mediated transepithelial transport of intact IgG in RPTECs is bidirectional and that it requires the formation of acidified intracellular compartments. With the use of double immunofluorescence, the internalized human IgG was marked in cytoplasm of RPTECs and colocalized with FcRn. These data define the mechanisms of FcRn-associated IgG transport in RPTEC monolayers. It was suggested that the intact pathway for human IgG transepithelial transport may avoid lysosomal degradation of IgG.

neonatal Fc receptor; immunoglobulin G transport; proximal tubule; immunoglobulin G homeostasis; mucosal immunity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AFTER BLOOD FILTRATION by glomeruli, filtered plasma proteins are reabsorbed via the endocytic pathway by renal proximal tubules. Albumin and beta 2-microglobulin, which are low-molecular-weight plasma proteins, and several hormones are reabsorbed via the receptor-mediated endocytic pathway by the proximal tubules (27). Reabsorption of these proteins via receptor-mediated endocytosis and their subsequent delivery to lysosomes for degradation in the proximal tubules may play important roles in protein and hormonal homeostasis (27). Under normal conditions, urinary protein includes ~40% albumin, 5-10% IgG, 5% immunoglobulin light chains, 3% IgA, and other proteins (6, 27). In glomerular diseases, a large amount of filtered plasma protein is followed by increased reabsorption in the proximal tubules and causes progression of chronic renal diseases (2, 24). Several studies suggest that reabsorption of albumin in the proximal tubular cells may be mediated by specific receptor-mediated endocytosis and that excessive reabsorption of albumin may induce expression of numerous proinflammatory genes (8, 36). Immunoglobulins are also present in urine under intact or impaired renal conditions. Some studies reported that immunoglobulin might induce tubular damage similar to other proteins (2, 38), but details of this mechanism are not clear.

In the kidneys, receptor-mediated transport of immunoglobulins has been studied in the polymeric immunoglobulin receptor, which transports polymeric IgA and IgM from the basolateral to the apical surface (3, 25). This receptor-mediated transport of polymeric IgA plays an important role in mucosal immunity of the urinary tract (31, 32). Other studies reported receptor-mediated endocytosis of the immunoglobulin light chain (4, 5), but the precise steps involved in IgG endocytosis and catabolism by the proximal tubular cells are unknown.

On the other hand, receptor-mediated endocytosis of IgG has been extensively studied in passive immunity from mothers to their young. This receptor is known as the neonatal Fc receptor (FcRn) and was initially identified in rodents as the receptor that mediates the transport of maternal immunoglobulins to the young via the neonatal intestine (17, 26). FcRn is associated with beta 2-microglobulin and is structurally homologous with the alpha -chain of the major histocompatibility complex class I molecule (28). One of the specific characteristics of FcRn is pH-dependent IgG binding, that is, high-affinity binding at acidic pH and weak or no binding at neutral pH. IgG is transported to the fetus or neonate across the intestinal epithelium or yolk sac in rodents (17, 26, 28) and across the placenta in humans (12, 19, 29, 30). It has been suggested that FcRn may play a critical role in passive immunity (12, 17, 19, 26, 28-30). More recently, several studies indicated that this receptor is implicated not only in transport of maternal IgG to the young for passive immunity, but also in maintenance of IgG homeostasis by recycling internalized IgG beyond the neonatal period (14, 15). Because internalized IgG that binds to FcRn is prevented from degradation in lysosomes, intact IgG may cross the epithelia.

Recently, Haymann et al. (16) reported that FcRn was expressed in the human renal glomerular epithelial cells and brush borders of the proximal tubular cells. They suggested that FcRn in proximal tubular cells may mediate endocytosis of IgG and play a role in the reabsorption of IgG from the tubular fluid, but the physiological function of FcRn has not been clarified. In the present study, the functional expression of FcRn and the endocytic pathway of IgG were examined in human RPTECs (hRPETCs).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and cell culture conditions. hRPTECs were purchased from Clonetics (San Diego, CA). Identity and purity of RPTECs were examined by staining with gamma -GTP and phase-contrast microscopy. RPTECs were cultured on collagen-coated plastic dishes and studied at passages 3-5 in renal epithelial cell growth medium (Iwaki Glass, Tokyo, Japan) containing 50 µg/ml gentamicin, 50 ng/ml amphotericin B, 10 µg/ml transferrin, 5 µg/ml insulin, 10 ng/ml human recombinant epidermal growth factor, 500 ng/ml epinephrine, and 0.5% fetal bovine serum (FBS). For transport studies, ~3 × 104 cells/cm2 were seeded on collagen-coated Transwell-Clear polyester membrane inserts (6.5 mm diameter, 0.4 µm pore size; Corning, Tokyo, Japan) to obtain polarized cell monolayers, as previously described in a rat kidney cell line (20). The presence of a continuous monolayer was routinely monitored by microscopic evaluation of all filters. The transepithelial electrical resistance measured by Millicell-ERS (Millipore, Bedford, MA) attained stable levels after 7 days (data not shown).

Human intestinal epithelial T84 cells were purchased from American Type Culture Collection (Manassas, VA) at passage 52 and maintained in a 1:1 mixture of F-12K and DMEM containing 5% FCS. Because U937 cells express a low amount of FcRn, we transfected a full-length cDNA encoding hFcRn into U937 cells and used them as positive controls. U937 cells transfected with or without the full-length cDNA encoding hFcRn were maintained in RPMI 1640 medium containing 10% FCS. Jurkat cells were also maintained in RPMI 1640 medium containing 10% FCS and used as negative controls.

RNA preparation and RT-PCR. Total RNA was isolated from RPTECs and T84 cells, peripheral blood mononuclear cells (PBMCs), and U937 cells using TRIzol (Life Technologies, Rockville, MD). Total RNA (2 µg) was converted to cDNA using oligo(dT) primers (Life Technologies) and reverse transcriptase (Superscript, Life Technologies). The single-strand cDNA product was denatured and amplified in a GeneAmp PCR system (model 9600, Perkin-Elmer, Norwalk, CT), with each set of primers chosen on the basis of the human FcRn (hFcRn), Fcgamma RI (CD64), Fcgamma RII (CD32), and Fcgamma RIII (CD16) sequences (10, 23, 30, 35). The regions amplified by each set of primers were as follows: 5'-ACT CCT GCC TTC TGG GTG TC-3' and 5'-GGT AGA AGG AGA AGG CGC TG-3' for FcRn nucleotides 255-807, 5'-CAG TGG AGA GTA CAG GTG CC-3' and 5'-CTC CTT GAA CAC CCA CCG AG-3' for CD16 nucleotides 282-393, and 5'-CCC AAA GGC TGT GCT GAA AC-3' and 5'-GTG GTT TGC TTG TGG GAT GG-3' for CD32 nucleotides 121-545. Two primers specific for CD64 cDNA were identical to those described previously (35). The PCR products were separated by electrophoresis on 2.0% agarose gels and visualized by ethidium bromide staining. The fidelity of the PCR products was also confirmed by nucleotide sequencing.

Affinity-purified rabbit anti-hFcRn antibody. Rabbit polyclonal anti-hFcRn antibody against the peptide was raised in New Zealand White rabbits. The peptide consisted of amino acids 112-125 of the alpha 2-domain of hFcRn plus an NH2-terminal Cys for conjugation (Sawady Technology, Tokyo, Japan) (29). Gene bank searches indicated that the sequence of the peptide was unique to hFcRn and showed no similarities to other molecules. Specific rabbit anti-hFcRn antibodies were isolated from immune serum by affinity purification on the peptide immobilized to a Sulfolink matrix (Pierce, Rockford, IL) according to the manufacturer's instructions. The antibodies worked well in Western blot analyses, immunoprecipitation, and immunostaining.

Immunoblot analysis. RPTECs, Jurkat cells, and U937 cells transfected with a full-length cDNA encoding hFcRn (U937-hFcRn) were extracted in 1% Nonidet P-40 in PBS. Protein concentrations in the extracts were determined by the bicinchoninic acid method (Pierce) with BSA standards. The extracts were resolved on 12.5% SDS-PAGE, transferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Yonezawa, Japan), and probed with a rabbit anti-hFcRn antibody (1 µg/ml) or preimmune serum. After incubation with a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cappel, ICN Pharmaceuticals, Aurora, OH), the signal was detected by an enhanced chemiluminescence system (ECL-plus, Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized by a luminescent image analyzer (model LAS-1000 plus, Fuji Film).

Immunoprecipitation. RPTECs, Jurkat cells, and U937-hFcRn cells (5 × 106) were lysed in 5 mg/ml 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in 50 mM phosphate buffer containing protease inhibitors (Complete, Boehringer Mannheim, Mannheim, Germany). After centrifugation, the supernatant was collected and precleared with 30 µl of protein G-Sepharose 4FF beads (Amersham Pharmacia Biotech). The lysates were then subjected to immunoprecipitation with a rabbit anti-hFcRn antibody or preimmune serum. The immunoprecipitates were collected on protein G-Sepharose 4B beads, washed with the lysis buffer, and extracted from the beads by boiling in 60 µl of sample buffer containing 2-mercaptoethanol. Then 20 µl of each sample were analyzed by SDS-PAGE (5-20% gradient gel; BC BioCraft, Tokyo, Japan), transferred onto the PVDF membrane, and probed with a goat anti-human beta 2-microglobulin antibody (Nippon Bio-Test Laboratories, Tokyo, Japan).

pH-dependent binding of cell lysates to human IgG-agarose. Affinity binding of hFcRn to human IgG-agarose was carried out as previously described with some modifications (12, 19, 22). Briefly, 6 × 106 RPTECs were lysed with 5 mg/ml 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate containing protease inhibitors in 50 mM phosphate buffer adjusted to pH 6.0 or 8.0. The solubilized total proteins were incubated with 60 µl of human IgG-agarose corresponding to 300 µg of IgG at 4°C for 12 h. The IgG-agarose beads were collected by centrifugation and washed with lysis buffer (pH 6.0 or 8.0). Bound proteins were eluted by boiling in sample buffer and analyzed by 12.5% SDS-PAGE under reducing conditions. For Western blot analysis, proteins were transferred onto the PVDF membrane and probed with a rabbit anti-hFcRn antibody and then with a goat anti-human beta 2-microglobulin antibody.

Human IgG binding assay. Biotinylated human IgG (Vector Laboratories, Burlingame, CA) binding assay was carried out as previously described (16) with some modifications. Briefly, RPTECs grown on 100-mm-diameter type I collagen-coated culture dishes (Iwaki Glass) were detached with 5 mM EDTA and resuspended in binding buffer (Hanks' balanced salt solution with 10 mM HEPES, pH 6.0 or 8.0, containing 0.1% BSA). The cells were pelleted, washed, and then resuspended in the binding buffer at 1 × 106 cells/ml. The cell suspension was mixed with biotinylated human IgG (10 µg/ml), with or without 1 mg/ml unlabeled human IgG. After incubation at 4°C for 4 h on a rotating mixer, the suspensions were spun down, and unbound ligands were removed by washing with the binding buffer at pH 6.0 or 8.0. The cells were then spun down and dissolved in 1% Nonidet P-40 in PBS. The same quantity of proteins was applied to 12.5% polyacrylamide denaturing gels, transferred onto the PVDF membrane, and probed with a horseradish peroxidase-conjugated avidin. The signal intensity of the bands was analyzed by a luminescent image analyzer (model LAS-1000 plus, Fuji Film).

Human IgG transport assay. Human IgG transport assay was performed as previously described (11) with some modifications (12, 20, 22). Briefly, RPTEC monolayers exhibiting stable electrical resistance were washed and equilibrated in Hanks' balanced salt solution with 10 mM HEPES, pH 7.4, containing 0.025% BSA. Biotinylated human IgG or IgY (Sigma, St. Louis, MO) was added at 100 µg/ml to the apical or basolateral chamber. Unlabeled IgG or IgY (10 mg/ml) was used as a competitive inhibitor. To evaluate the effects of pharmacological agents that interfere with cell trafficking, RPTEC monolayers were pretreated with 0.1 µM bafilomycin A1 (Sigma), which alkalinizes endocytic vesicles by specifically inhibiting the vacuolar proton pump (11, 13). RPTECs were incubated with ligands at 37°C or 4°C, and contralateral chamber medium was collected at various times. Transported proteins were concentrated (Centricon YM-100, Millipore, Bedford, MA) and analyzed by SDS-PAGE and ligand blot after reduction with 2-mercaptoethanol. The signal intensity of the bands was compared using the luminescent image analyzer (model LAS-1000 plus, Fuji Film) and measured against control biotinylated human IgG (12.5 ng of biotinylated human IgG standard).

Immunofluorescence. RPTECs grown on glass coverslips were incubated for 1 h at 4°C in binding buffer (0.05% BSA and Hanks' balanced salt solution with 10 mM HEPES, pH 6.0 or 8.0) containing 1 mg/ml human IgG. The cells were then washed with PBS at pH 6.0 or 8.0 to remove nonbound human IgG. Thereafter, the cells were incubated at 37°C for another 30 min to allow internalization of the bound human IgG in binding buffer (pH 6.0 or 8.0). After internalization, the cells were washed, fixed with 2% formaldehyde and 4% sucrose in PBS, and then permeabilized with 0.3% Triton X-100 in PBS. After the cells were blocked with 2% BSA, 2% FCS, and 0.2% fish gelatin in PBS for 30 min, FITC-conjugated goat anti-human IgG antibody (1:100; Jackson ImmunoResearch, West Grove, PA) was applied, and the cells were incubated at room temperature for 1 h. Colocalization of the internalized human IgG with hFcRn was detected using rabbit anti-hFcRn antibody (1:50) and rhodamine-conjugated goat anti-rabbit IgG antibody (1:100; Cappel, ICN Pharmaceuticals). To detect the cell surface expression and steady-state distribution of hFcRn, RPTECs grown on glass coverslips were fixed with or without permeabilization using 0.3% Triton X-100 in PBS. Staining procedures to detect hFcRn were the same as those described above. The fixed cells were mounted in Immunon (Shandon, Pittsburgh, PA) and then viewed using a confocal microscope (model MRC-1024, Bio-Rad).

These experiments were reproduced at least three times by independent studies.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression and physical association of hRcRn and beta 2-microglobulin in RPTECs. Specific mRNA for the FcRn was expressed in RPTECs by RT-PCR (Fig. 1A). PCR products of the expected size (553 bp) were obtained from RPTECs and T84 cells, a human intestinal epithelial cell line expressing functional hFcRn (positive control). To further confirm whether the detected hFcRn mRNA was translated to the detectable protein, Western blot was performed using RPTECs, Jurkat cells, and U937-hFcRn cell extracts. As shown in Fig. 1B, a single specific band of ~45 kDa was detected in RPTECs (lane 1), as in U937-hFcRn cells (lane 3) but not in Jurkat cells (lane 2), using rabbit anti-hFcRn antibody. These bands were not detected with the preimmune serum (lanes 4-6). Because hFcRn associates with beta 2-microglobulin, immunoprecipitation of the cell lysates using the same antibody was performed as Western blot analysis (Fig. 1C). Approximately 12-kDa bands were detected in RPTECs (lane 3), U937-hFcRn cells (lane 1), and purified human beta 2-microglobulin (lanes 4 and 8), but not in Jurkat cells (lane 2). These bands were not detected when the cells were immunoprecipitated with the preimmune serum (lanes 5-7). To determine whether FcRn expressed in RPTECs preserves specific pH-dependent binding capacity for IgG, the cell lysates incubated with human IgG-agarose at pH 6.0 or 8.0 were analyzed by Western blot. As shown in Fig. 1D, hFcRn preferentially bound to human IgG-agarose at pH 6.0 (lane 1) and coprecipitated with beta 2-microglobulin (lane 3), but the binding was significantly reduced at pH 8.0 (lane 2). It was assumed that two other bands of lanes 3 and 4 were nonspecific reactions with heavy and light chains of human IgG. hFcRn protein was expressed in RPTECs and physically associated with beta 2-microglobulin, and it preserved specific pH-dependent IgG binding.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1.   Human neonatal Fc receptor (hFcRn) expression and physical association with beta 2-microglobulin in renal proximal tubular endothelial cells (RPTECs). A: RT-PCR analysis of T84 cells (lanes 1 and 2) and RPTECs (lanes 3 and 4). PCR was performed with primers specific for hFcRn cDNA, and products were analyzed on ethidium bromide-stained 2.0% agarose gel. Lanes 2 and 4, negative control (without reverse transcriptase); lane 5, negative control (water as template); lane M, molecular base marker (100-bp ladder). B: immunoblot of cell extracts from RPTECs (lanes 1 and 4), Jurkat cells (lanes 2 and 5), and U937-hFcRn cells (lanes 3 and 6) probed with a rabbit anti-hFcRn antibody (lanes 1, 2, and 3, respectively) or preimmune serum (lanes 4, 5, and 6, respectively). Total protein was loaded at 20 µg/lane. C: immunoprecipitates with rabbit anti-hFcRn antibody (lanes 1-3) or preimmune serum (lanes 5-7). Proteins transferred onto the polyvinylidene difluoride (PVDF) membrane were probed with a goat anti-human beta 2-microglobulin antibody. Lanes 1 and 5, U937-hFcRn cells; lanes 2 and 6, Jurkat cells; lanes 3 and 7, RPTECs; lanes 4 and 8, 0.3 ng of human beta 2-microglobulin (beta 2-MG). D: Western blot analysis of cell lysates from RPTECs incubated with human IgG-agarose at pH 6.0 or 8.0. Membrane was probed with a rabbit anti-hFcRn antibody (lanes 1 and 2) or reprobed with a goat anti-human beta 2-microglobulin antibody (lanes 3 and 4).

Cell surface and intracellular expression of hFcRn in RPTECs. Cell surface and intracellular expression of hFcRn was detected with the specific antibody (Fig. 2, A and B). No staining was observed when RPTECs were incubated with preimmune serum and secondary antibodies (Fig. 2, C and D). hFcRn was constitutively present on the plasma membrane and in the cytoplasm of RPTECs.


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2.   Cell surface expression and steady-state distribution of hFcRn in RPTECs. RPTECs grown on glass coverslips were fixed, either permeabilized (B and D) or not (A and C), and stained with a rabbit anti-hFcRn antibody and then with a rhodamine-labeled secondary antibody. Expression of hFcRn was detected at the cell surface (A) and the cytoplasm (B). No labeling was observed when RPTECs were stained with preimmune serum and secondary antibody (C and D).

No detection of Fcgamma RI, Fcgamma RII, and Fcgamma RIII transcripts in RPTECs. As shown in Fig. 3, PCR products of the expected size were detected as follows: 112-bp fragment specific for CD 16 in the PBMCs (lane 1) and 425-bp fragment specific for CD 32 (lane 6) and 600- and 880-bp fragments specific for CD64 (lane 6) in U937 cells but not in RPTECs (lanes 3 and 8). RT-PCR amplification of human glyceraldehyde-3-phosphate dehydrogenase provides an internal control for each reaction (lanes 1, 3, 6, and 8). There was no genomic DNA contamination, because RT-PCR performed on each RNA without reverse transcriptase yielded negative results (lanes 2, 4, 7, and 9).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.   Gene expression of Fcgamma RI (CD64), Fcgamma RII (CD32), and Fcgamma RIII (CD16). Total RNA from peripheral blood mononuclear cells (PBMCs; lanes 1 and 2, positive control), U937 cells (lanes 6 and 7, positive control), and RPTECs (lanes 3, 4, 8, and 9) were incubated with an oligo(dT) primer with (lanes 1, 3, 6, and 8) or without (lanes 2, 4, 7, and 9) RT. PCR was performed with primers specific for each Fcgamma R sequence or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR products were analyzed on an ethidium bromide-stained 2.0% agarose gel. Lanes 5 and 10, PCR negative control (water as template); lane M, molecular base marker (100-bp ladder).

Specific pH-dependent human IgG binding to the cell surface of RPTECs. Biotinylated human IgG could bind to RPTECs in a specific manner at pH 6.0, and binding of biotinylated human IgG was significantly reduced in the presence of excess amounts of unlabeled IgG at pH 6.0 (Fig. 4). In contrast, binding levels of biotinylated human IgG were much lower at pH 8.0. The specific pH-dependent IgG binding proteins were expressed on the plasma membrane of RPTECs.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   pH-dependent binding of IgG to the cell surface of RPTECs. Percent inhibition of biotinylated IgG binding at pH 6.0 and 8.0. Triplicate assays were performed in the absence (-, solid bars) or presence (+, open bars) of a 100-fold excess of unlabeled IgG to assess specificity of the binding. Result is typical of Western blot analysis as a heavy chain. Values (means ± SD) are expressed as percentage of binding in the absence of unlabeled IgG at pH 6.0.

Receptor-mediated transcytosis of human IgG in RPTECs. FcRn-dependent IgG transport in RPTEC monolayers by transepithelial flux of biotinylated human IgG was investigated using Transwell inserts. Biotinylated human IgG was added to the apical or basolateral chamber of the cell culture inserts at 4°C or 37°C. Intact human IgG was transported in both directions Fig. 5. Transport of human IgG was detected in monolayers incubated at 37°C but not in those incubated at 4°C (data not shown). Chicken IgY was not transported at detectable levels in either direction (data not shown). To further confirm whether this receptor-mediated transcytosis was specific for IgG, we analyzed biotinylated human IgG transport with excess unlabeled human IgG or chicken IgY as the competitive inhibitor. Bidirectional transcytosis of biotinylated human IgG was significantly reduced in the presence of excess unlabeled IgG (Fig. 5). In contrast, excess chicken IgY did not compete with biotinylated human IgG (Fig. 5). The receptor-mediated transport was specific for IgG. To evaluate the effects of pharmacological agents that interfere with acidification of the endosomes, RPTEC monolayers were pretreated with bafilomycin A1, a specific inhibitor of H+-ATPase. Bidirectional transcytosis of biotinylated human IgG was significantly reduced by pretreatment with bafilomycin A1 (Fig. 5), suggesting that the receptor-mediated transepithelial transport of IgG in RPTECs requires acidified intracellular compartments.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   Receptor-mediated transcytosis of IgG across RPTEC monolayers. Transcytosis of biotinylated human IgG was detected in RPTEC monolayers at 37°C. Bidirectional transcytosis was significantly reduced in monolayers pretreated with 0.1 µM bafilomycin A1 and in the presence of a 100-fold excess of unlabeled human IgG but not chicken IgY. A and B: apical-to-basolateral and basolateral-to-apical transport, respectively. Values are means ± SD of 4 different experiments.

Colocalization of the hFcRn and internalized human IgG in RPTECs. To identify the pH-dependent IgG binding protein as hFcRn, we visualized the localization of hFcRn and internalization of human IgG in RPTECs. Human IgG bound to the RPTEC cell surface at pH 6.0 was internalized after incubation at 37°C (Fig. 6A). hFcRn was also detected in cytoplasm with a rabbit anti-hFcRn antibody (Fig. 6B). Merging of Fig. 6, A and B, showed extensive colocalization of hFcRn and human IgG (Fig. 6C). In contrast, internalized IgG was not detected in RPTECs when preincubated with human IgG at pH 8.0 (Fig. 6D), despite hFcRn expression (Fig. 6E). hFcRn mediated transcytosis of human IgG in RPTECs.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Colocalization of hFcRn and internalized IgG in RPTECs. RPTECs grown on glass coverslips were incubated at 4°C with human IgG in pH 6.0 or 8.0 solution. After cells were washed and incubated at 37°C, internalized human IgG was detected with an FITC-conjugated goat anti-human IgG antibody at pH 6.0 (A) and pH 8.0 (D). hFcRn was detected with a rabbit anti-hFcRn antibody at pH 6.0 (B) and pH 8.0 (E). Merging of A and B shows colocalization of internalized human IgG and hFcRn (C). F: merging of D and E. Colocalization of human IgG (D) and hFcRn (E) was not observed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The endocytic function of RPTECs has been studied with various proteins that are present in the urine (27, 33). Albumin, the most prominent protein in glomerular filtrate, is reabsorbed by proximal tubules via specific receptor-mediated endocytosis (8, 21). Although the reabsorption of albumin prevents the loss of large amounts of this major plasma protein via the urine, excess reabsorption of albumin may be a factor for the development and progression of chronic renal diseases (8, 36). Thus in the kidney there appears to be a limit on the amount of protein that can be reabsorbed and catabolized at the greatly increased filtered loads found in renal diseases. On the other hand, immunoglobulins such as IgG, IgA, and their fragments, including free light chains and Fc portions of IgG heavy chains, have also been found in the urine (27). Similar to other proteins, absorption of these molecules seems to be regulated by the endocytosis pathway in the proximal tubules, but the mechanisms are not fully understood (1, 34). Therefore, the clarification of the endocytosis pathway of these proteins in the proximal tubular epithelial cells is important physiologically and pathophysiologically.

In the present study, we examined whether FcRn, which is substantially involved in IgG transport in other tissues (15), is functionally expressed in hRPTECs and mediates endocytosis of IgG. FcRn was actually expressed in RPTECs associated with beta 2-microglobulin and preserved pH-dependent binding with IgG. In a steady state, FcRn in RPTECs was distributed on the cell surface and in the cytoplasm, indicating an FcRn-IgG interaction in RPTECs on the cell surface and in endocytic vesicles, in agreement with recent studies (11, 12, 20). Indeed, human IgG could bind to the cell surface of the RPTECs in a pH-dependent manner with a high affinity at acidic pH and with a low or no affinity at neutral pH. The preferential binding of IgG to RPTECs observed at pH 6.0 is consistent with the presence of FcRn on the cell surface. Furthermore, IgG was transported across the RPTEC monolayers bidirectionally, which required endosomal acidification. On the other hand, typical Fcgamma receptors such as CD16 (Fcgamma RIII), CD32 (Fcgamma RII), and CD64 (Fcgamma RI) were not expressed in RPTECs at the mRNA level detected by RT-PCR. It was suggested that transepithelial transport of human IgG in RPTECs is mediated by FcRn. Colocalization of FcRn and the internalized human IgG shown by double immunofluorescence also supports the transepithelial transport of IgG in RPTECs by FcRn.

FcRn has been identified and characterized in various organs such as the small intestine, liver, mammary gland, placenta, and yolk sac (11, 29, 30). This receptor is functionally expressed beyond the neonatal period and is potentially relevant to other postnatal functions, including the protection of IgG from catabolism (15). In all studies, multiple functions have been identified, but the molecular details of functions in distinct cellular environments are not fully understood (15). It is suggested that there are two important functions of this receptor. First, FcRn transports maternal IgG to the fetus or neonate. In rodents, maternal IgG transport occurs in the yolk sac or neonatal intestine, whereas in humans, essentially all IgGs are transferred prenatally across the placenta (29, 30). Second, FcRn is responsible for the maintenance of serum IgG levels by protecting plasma IgG from catabolism (14). These functions are supported by the fact that IgGs are salvaged from lysosomal degradation when they bind to FcRn, while IgG that does not bind to FcRn is destined for degradation in lysosomes (15). Because IgG is one of the most quantitatively important plasma proteins in the urine (6, 9) and accounts for most urinary immunoglobulins, we propose that IgG transport from the apical to the basolateral surface via FcRn in RPTECs reveals reabsorption of IgG from tubular fluid and that it may play an important role in IgG homeostasis (14).

Abbate et al. (2) reported that interstitial cellular infiltration developed at or near tubules containing intracellular IgG or luminal casts under high levels of urinary protein excretion observed in different models of proteinuric nephropathies. In cultured proximal tubular cells, IgG stimulates synthesis of endothelin-1 and RANTES production, which may play a role in the interstitial inflammatory reaction (37, 38). As discussed above, excess reabsorption of IgG may be an important factor in the development and progression of chronic renal diseases.

IgG transport from the basolateral to the apical surface via FcRn in RPTECs suggested that the proximal tubular epithelial cells may physiologically transport IgG to the mucosal surface. In a previous study using a human intestinal epithelial cell line (11), it was revealed that FcRn has important effects on IgG-mediated mucosal immunity and host defense in adult intestines. It has also been demonstrated that IgG is present in secretions of the human mucous membranes such as oral mucosa, lung, intestine, and genitourinary tract (7, 11, 18, 32). In the kidney, the mucosal immune response has been intensively studied in urinary tract infections (31). For instance, urine from patients with urinary tract infections often contains antibodies against the infecting strain, particularly secretory IgA (32). Polymeric immunoglobulin receptor, which is produced by secretory epithelial cells, transports polymeric IgA from the basolateral to the apical surface, suggesting an important role in mucosal immunity of the urinary tract by secretion of secretory IgA (3, 25). Luminal secreted IgG may be of local origin and transported selectively across mucosal barriers in the same way as IgA (7, 18).

In summary, we demonstrated that IgG is transported across RPTEC monolayers by FcRn, which requires endosomal acidification in binding of IgG and prevents lysosomal degradation of IgG. Our studies not only identified receptor-mediated IgG transport in RPTECs but also raised the possibility that FcRn may have a role in the reabsorption of IgG from tubular fluid and in mucosal immunity of the urinary tract because of the bidirectional IgG transport in RPTECs. Further investigation is required to determine whether this receptor has functional relevance to these hypotheses in vivo.


    ACKNOWLEDGEMENTS

The authors are grateful to Drs. I. Shirato and S. Horikoshi for helpful discussions and critical reading of the manuscript, Drs. H. Matsuda, K. Kawamoto, and A. Yoshino for helpful discussions and technical support, and T. Shibata and T. Shigihara for excellent technical assistance.


    FOOTNOTES

This work was supported in part by a grant from the Japanese Ministry of Education, Science, and Culture (Tokyo, Japan).

Address for reprint requests and other correspondence: Y. Tomino, Div. of Nephrology, Dept. of Internal Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan (E-mail: yasu{at}med.juntendo.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published August 15, 2001; 10.1152/ajprenal.00164.2001

Received 25 May 2001; accepted in final form 12 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aarli, A, Matre R, and Thunold S. IgG Fc receptors on epithelial cells of distal tubuli and on endothelial cells in human kidney. Int Arch Allergy Appl Immunol 95: 64-69, 1991[ISI][Medline].

2.   Abbate, M, Zoja C, Corna D, Capitanio M, Bertani T, and Remuzzi G. In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation. J Am Soc Nephrol 9: 1213-1224, 1998[Abstract].

3.   Abramowsky, CR, and Swinehart GL. Secretory immune responses in human kidneys. Am J Pathol 125: 571-577, 1986[Abstract].

4.   Batuman, V, and Guan S. Receptor-mediated endocytosis of immunoglobulin light chains by renal proximal tubule cells. Am J Physiol Renal Physiol 272: F521-F530, 1997[Abstract/Free Full Text].

5.   Batuman, V, Verroust PJ, Navar GL, Kaysen JH, Goda FO, Campbell WC, Simon E, Pontillon F, Lyles M, Bruno J, and Hammond TG. Myeloma light chains are ligands for cubilin (gp280). Am J Physiol Renal Physiol 275: F246-F254, 1998[Abstract/Free Full Text].

6.   Berggard, I. Plasma proteins in normal human urine. In: Proteins in Normal and Pathological Urine, edited by Manuel Y, Revillard JP, and Betuel H.. New York: Karger, 1970, p. 7-19.

7.   Berneman, A, Belec L, Fischetti VA, and Bouvet JP. The specificity patterns of human immunoglobulin G antibodies in serum differ from those in autologous secretions. Infect Immun 66: 4163-4168, 1998[Abstract/Free Full Text].

8.   Birn, H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, Willnow TE, Moestrup SK, and Christensen EI. Cubilin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 105: 1353-1361, 2000[Abstract/Free Full Text].

9.   Bohle, A, Oliveira de Cavalcanti V, and Laberke HG. Is there any tubular secretion of protein? Clin Nephrol 29: 28-34, 1988[ISI][Medline].

10.   Brooks, DG, Qiu WQ, Luster AD, and Ravetch JV. Structure and expression of human IgG FcRII (CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes. J Exp Med 170: 1369-1385, 1989[Abstract].

11.   Dickinson, BL, Badizadegan K, Wu Z, Ahouse JC, Zhu X, Simister NE, Blumberg RS, and Lencer WI. Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest 104: 903-911, 1999[Abstract/Free Full Text].

12.   Ellinger, I, Schwab M, Stefanescu A, Hunziker W, and Fuchs R. IgG transport across trophoblast-derived BeWo cells: a model system to study IgG transport in the placenta. Eur J Immunol 29: 733-744, 1999[ISI][Medline].

13.   Gekle, M, Mildenberger S, Freudinger R, and Silbernagl S. Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells. Am J Physiol Renal Fluid Electrolyte Physiol 268: F899-F906, 1995[Abstract/Free Full Text].

14.   Ghetie, V, Hubbard JG, Kim JK, Tsen MF, Lee Y, and Ward ES. Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur J Immunol 26: 690-696, 1996[ISI][Medline].

15.   Ghetie, V, and Ward ES. Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annu Rev Immunol 18: 739-766, 2000[ISI][Medline].

16.   Haymann, JP, Levraud JP, Bouet S, Kappes V, Hagege J, Nguyen G, Xu Y, Rondeau E, and Sraer JD. Characterization and localization of the neonatal Fc receptor in adult human kidney. J Am Soc Nephrol 11: 632-639, 2000[Abstract/Free Full Text].

17.   Jones, EA, and Waldmann TA. The mechanism of intestinal uptake and transcellular transport of IgG in the neonatal rat. J Clin Invest 51: 2916-2927, 1972[ISI][Medline].

18.   Kozlowski, PA, Cu-Uvin S, Neutra MR, and Flanigan TP. Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect Immun 65: 1387-1394, 1997[Abstract].

19.   Leach, JL, Sedmak DD, Osborne JM, Rahill B, Lairmore MD, and Anderson CL. Isolation from human placenta of the IgG transporter, FcRn, and localization to the syncytiotrophoblast: implications for maternal-fetal antibody transport. J Immunol 157: 3317-3322, 1996[Abstract].

20.   McCarthy, KM, Yoong Y, and Simister NE. Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia. J Cell Sci 113: 1277-1285, 2000[Abstract/Free Full Text].

21.   Park, CH, and Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit. J Clin Invest 73: 767-777, 1984[ISI][Medline].

22.   Praetor, A, Ellinger I, and Hunziker W. Intracellular traffic of the MHC class I-like IgG Fc receptor, FcRn, expressed in epithelial MDCK cells. J Cell Sci 112: 2291-2299, 1999[Abstract/Free Full Text].

23.   Ravetch, JV, and Perussia B. Alternative membrane forms of Fcgamma RIII (CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions. J Exp Med 170: 481-497, 1989[Abstract].

24.   Remmuzi, G, and Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 339: 1448-1456, 1998[Free Full Text].

25.   Rice, JC, Spence JS, Megyesi J, Goldblum RM, and Safirstein RL. Expression of the polymeric immunoglobulin receptor and excretion of secretory IgA in the postischemic kidney. Am J Physiol Renal Physiol 276: F666-F673, 1999[Abstract/Free Full Text].

26.   Rodewald, R. Distribution of immunoglobulin G receptors in the small intestine of the young rat. J Cell Biol 85: 18-32, 1980[Abstract].

27.   Rosengerg, ME, and Hostetter TH. Proteinurea. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW, and Giebish G.. New York: Raven, 1992, p. 3005-3061.

28.   Simister, NE, and Mostov KE. An Fc receptor structurally related to MHC class I antigens. Nature 337: 184-187, 1989[ISI][Medline].

29.   Simister, NE, Story CM, Chen HL, and Hunt JS. An IgG-transporting Fc receptor expressed in the syncytiotrophoblast of human placenta. Eur J Immunol 26: 1527-1531, 1996[ISI][Medline].

30.   Story, CM, Mikulska JE, and Simister NE. A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus. J Exp Med 180: 2377-2381, 1994[Abstract].

31.   Svanborg, C, de Man P, and Sandberg T. Renal involvement in urinary tract infection. Kidney Int 39: 541-549, 1991[ISI][Medline].

32.   Svanborg, C, and Svennerholm AM. Secretory immunoglobulin A and G antibodies prevent adhesion of Escherichia coli to human urinary tract epithelial cells. Infect Immun 22: 790-797, 1978[ISI][Medline].

33.   Trowbridge, IS, Collawn JF, and Hopkins CR. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 9: 129-161, 1993[ISI].

34.   Tuijnman, WB, Van Wichen DF, and Schuurman HJ. Tissue distribution of human IgG Fc receptors CD16, CD32 and CD64: an immunohistochemical study. APMIS 101: 319-329, 1993[ISI][Medline].

35.   Van de Winkel, JG, de Wit TP, Ernst LK, Capel PJ, and Ceuppens JL. Molecular basis for a familial defect in phagocyte expression of IgG receptor I (CD64). J Immunol 154: 2896-2903, 1995[Abstract/Free Full Text].

36.   Wang, Y, Rangan GK, Tay YC, Wang Y, and Harris DC. Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor-kappa B in proximal tubule cells. J Am Soc Nephrol 10: 1204-1213, 1999[Abstract/Free Full Text].

37.   Zoja, C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, and Remuzzi G. Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation. Kidney Int 53: 1608-1615, 1998[ISI][Medline].

38.   Zoja, C, Morigi M, Figliuzzi M, Bruzzi I, Oldroyd S, Benigni A, Ronco P, and Remuzzi G. Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 26: 934-941, 1995[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 282(2):F358-F365
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society