1 Division of Nephrology, Department of Internal Medicine, and 2 Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan
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ABSTRACT |
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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
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
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INTRODUCTION |
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AFTER BLOOD
FILTRATION by glomeruli, filtered plasma proteins are reabsorbed
via the endocytic pathway by renal proximal tubules. Albumin and
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
2-microglobulin and is structurally homologous with the
-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).
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MATERIALS AND METHODS |
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Cells and cell culture conditions.
hRPTECs were purchased from Clonetics (San Diego, CA). Identity and
purity of RPTECs were examined by staining with -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).
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), FcRI (CD64), Fc
RII (CD32), and
Fc
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 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 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 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 |
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Expression and physical association of hRcRn and
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
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
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
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
2-microglobulin,
and it preserved specific pH-dependent IgG binding.
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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.
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No detection of FcRI, Fc
RII, and Fc
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).
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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.
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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.
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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.
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DISCUSSION |
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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 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 Fc
receptors
such as CD16 (Fc
RIII), CD32 (Fc
RII), and CD64 (Fc
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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