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Pathology Research Laboratory, Massachusetts General Hospital, Harvard
Medical School, Charlestown, Massachusetts.
Program in Membrane Biology, Massachusetts General Hospital, Harvard
Medical School, Charlestown, Massachusetts.
Correspondence to Dr. Michele Marinò, Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy. Phone: +39-050-544723; Fax: +39-050-578772; E-mail: m.marino{at}endoc.med.unipi.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reabsorption of RBP-retinol prevents urinary loss of vitamin A (retinol) and provides a way for it to be returned to the circulation, which is considered to be important in vitamin A homeostasis (1). However, the mechanisms by which vitamin A is recycled are uncertain. Christensen et al. (6) proposed that recycling occurs in two steps: first, the RBP-retinol complex is transported by megalin to lysosomes, where RBP is degraded, thus liberating retinol; second, the released retinol combines with newly synthesized RBP near the basolateral surface, where the RBP-retinol complex is secreted into the bloodstream. Because most ligands that are endocytosed by megalin are transported to lysosomes (11,12,13,14,15), the pathway proposed by Christensen is plausible. However, we recently showed that certain megalin ligands are transported intact across cells by transcytosis (16). In the present study, by the use of polarized immortalized rat renal proximal tubule (IRPT) cells, we provide evidence that RBP transepithelial passage after megalin-mediated endocytosis occurs by transcytosis, thereby providing an alternative explanation for recycling of RBP-retinol from the glomerular filtrate to the blood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human megalin was immunoaffinity purified by a previously described procedure (18), from fresh kidney specimens, obtained at autopsy at the Department of Pathology of the Massachusetts General Hospital from patients without renal disease. Ethylenediaminetetraacetate was used during megalin preparation to eliminate any contaminating receptor-associated protein (RAP).
RAP was used as a glutathione-S-transferase (GST) fusion protein.
DH5
bacteria harboring the pGEX-RAP expression construct were kindly
provided by Dr. Joachim Herz (University of Texas Southern Medical Center,
Dallas, TX). The production of RAP-GST and GST was performed as described
(19). Biotin-labeled bovine
serum albumin (BSA) and human lysozyme were obtained from Sigma. GST and
lysozyme were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin.
A mouse monoclonal antibody, designated 1H2, which reacts with megalin ectodomain epitopes in the second cluster of ligand binding repeats, was previously described (18). A rabbit antibody against human RBP was purchased from Dako Corporation (Carpinteria, CA). Alkaline phosphatase (ALP) conjugated streptavidin was from Vector (Burlingame, CA). Horseradish peroxidase (HRP) conjugated streptavidin was purchased from Amersham (Arlington Heights, IL). HRP-conjugated and ALP-conjugated goat anti-rabbit IgG were purchased from Bio Rad (Hercules, CA).
Solid Phase Binding Assays
Ninety-six-well microtiter plates were coated overnight at 4°C with 100
µl of unlabeled human megalin at a concentration of 100 µg/ml in
phosphate-buffered saline (PBS). Wells were blocked with BSA, washed with
Tris-buffered saline (TBS) containing 0.05% Tween-20, and incubated for 1 h at
room temperature with various concentrations of 125I-labeled RBP,
in TBS, 5 mM CaCl2, 0.5 mM MgCl2, 0.5% BSA, 0.05%
Tween-20. 125I-labeled RBP was added to the wells alone or in the
presence of an excess of unlabeled RBP at a concentration of 100 µg/ml. To
detect bound 125I-labeled RBP, wells were washed four times with
TBS, 0.05% Tween-20, and cut out, and radioactivity was measured with a
counter.
Cell Cultures
A rat immortalized renal proximal tubule cell line (IRPT cells) was
established as described previously
(20). These cells have
previously been shown to express megalin but not the LDL receptor-related
protein (LRP)
(20,21).
Therefore, RAP, which can inhibit ligand binding to both megalin and LRP, can
be considered in IRPT cells a specific megalin inhibitor. IRPT cells were
cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum.
Transcytosis Experiments
IRPT cells were cultured in high-density, large-pore (3 µm) filters in
cell culture inserts (Becton Dickinson, Mountain View, CA) placed in 24-well
plates, as described previously
(16). These devices allow
polarization of the cells and formation of tight junctions and make it
possible to trace passage of molecules across the cell layer, from the upper
(insert) to the lower (cell culture well) chamber. Cells were used at complete
confluence. The mean number of cells at confluence was 5.1 x
104 cells/well, and the mean amount of protein in cell lysates was
approximately 4.96 µg/well, as assessed using a commercial kit (Bio Rad).
The tightness of cell layers was assessed by measuring the transepithelial
electrical resistance (TER) and by measuring the paracellular transport of
3H-mannitol.
TER was measured using a Millicell-ERS conductivity meter from Millipore
(Bedford, MA), according to the manufacturer's instructions. As a blank, TER
was measured in filters without cells. TER values were expressed as Ohm
(
) x cm2.
Transport of 3H-mannitol was measured in IRPT cells as follows. Four µCi of 3H-mannitol (NEN Life Science Products) were added to the upper chamber, in a volume of 500 µl, in complete cell culture medium containing unlabeled mannitol (1 mM). The lower chamber was rinsed with 1 ml of medium without 3H-mannitol. Aliquots of the medium from the lower chamber were collected at various time points. Radioactivity was measured with a ß counter. Results were compared with those obtained in filters without cells.
To study transcytosis, we incubated confluent cells on filters at 37°C with various concentrations of either unlabeled or biotin-labeled RBP, in Coon's F12 medium, containing 5 mM CaCl2, 0.5 mM MgCl2, 0.5% ovalbumin (OVA). Biotin-labeled GST, biotin-labeled BSA, or unlabeled lysozyme was used as control. RBP or controls were added to the upper chamber in a volume of 200 µl, and the lower chamber was rinsed with 200 µl of buffer without ligands. After 6 h, fluids from the lower chambers were collected and RBP was measured by enzyme-linked immunosorbent assay (ELISA) or Western blotting or was subjected to immunoprecipitation. In addition, the material collected from the lower chamber was analyzed by nondenaturing PAGE, as detailed above. In megalin inhibition experiments, RBP was added to the cells together with the megalin competitors RAP-GST (200 µg/ml) or 1H2 (200 µg/ml) or, as controls, with GST (200 µg/ml) or normal mouse IgG (200 µg/ml). In certain experiments, incubations were performed at 4°C, at which temperature transcytosis is inhibited (22).
In certain experiments, transport of RBP across IRPT cells and transport of
control proteins was measured simultaneously. In a first set of experiments,
we measured transport of 3H-mannitol and 125I-RBP. IRPT
cells on permeable filters were incubated at 37°C with
125I-labeled RBP (0.5 µM) and 3H-mannitol (0.5
µM), which were added together to the upper chamber in 500 µl of Coon's
F12 medium, containing 5 mM CaCl2, 0.5 mM MgCl2, 0.5%
OVA. The lower chamber was rinsed with 1 ml of buffer without either
125I-labeled RBP or 3H-mannitol. After 6 h, the buffer
from the lower chamber was collected and radioactivity was measured with both
a and a ß counter.
In a second set of experiments we measured simultaneously transport of biotin-labeled RBP and biotin-labeled lysozyme. IRPT cells on filters were incubated for 6 h at 37°C with biotin-labeled RBP and biotin-labeled lysozyme (both at a 5-µM concentration), added together to the upper chamber. Fluids collected from the lower chamber were analyzed by Western blotting.
Immunoprecipitation Experiments
Samples collected from the lower chamber in biotin-labeled RBP transcytosis
experiments with IRPT cells were precleared with protein A agarose beads
(Pharmacia Biotech, Piscataway, NJ) coupled with normal rabbit IgG overnight
at 4°C. Beads were spun down and supernatants were collected and incubated
overnight at 4°C with the anti-RBP antibody (5 µl per sample) and with
50 µl of protein A agarose beads. After extensive washing, beads were
resuspended in nonreducing Laemmli buffer and spun down, and the supernatant
was subjected to SDS-PAGE and Western blotting, performed as described
below.
Immunofluorescence Staining of RBP Analyzed by Confocal
Microscopy
Unlabeled RBP (50 µg/ml) or binding buffer that lacked RBP was added to
the upper chamber of IRPT cells that were cultured until confluence on
permeable filters. Cells were then incubated at 37°C for 10 or 60 min,
after which the filters were washed three times with ice-cold PBS and fixed
with 4% paraformaldehyde-L-lysine-sodium periodate, 5% sucrose, in phosphate
buffer (0.1 M). The filters were treated with Triton-X100 to permeabilize
cells, mounted on glass coverslips, washed, and incubated with the rabbit
anti-RBP antibody (1:100), followed by FITC-conjugated goat anti-rabbit IgG
(Cappel, Durham, NC; 1:1000). Slides were examined by confocal microscopy.
Degradation Experiments
IRPT cells were cultured on plastic in 24-well plates until 80 to 100%
confluence was reached. The mean number of cells used in these experiments was
3.47 x 105 cells/well. The mean amount of protein in cell
lysates was approximately 35 µg/well. Cells, either untreated or treated
for 30 min at 37°C with chloroquine (50 µM in binding buffer), were
incubated at 37°C with 125I-RBP (20 µg/ml in 200 µl of
Coon's F12 medium, 5 mM CaCl2, 0.5 mM MgCl2, 0.5% OVA).
After 6 h, the binding buffer was collected and subjected to precipitation
with 10% trichloroacetic acid (TCA). Radioactivity was assessed with a
counter in the material nonprecipitable with TCA, as a measure of RBP
degradation. In megalin competition experiments, 125I-RBP was added
to the cells together with RAP-GST (200 µg/ml) or 1H2 (200 µg/ml) or, as
controls, with GST (200 µg/ml) or normal mouse IgG (200 µg/ml).
To calculate how much of RBP that was internalized by IRPT cells underwent
transcytosis or degradation, we performed experiments in which transcytosis
and degradation of RBP were measured simultaneously. IRPT cells were cultured
on filters as reported above and incubated at 37°C with
125I-labeled-RBP (20 µg/ml) added to the upper chamber in 500
µl of Coon's F12 medium, 5 mM CaCl2, 0.5 mM MgCl2,
0.5% OVA, whereas the lower chamber was rinsed with 1 ml of buffer without
RBP. After 6 h, fluids from the upper and lower chambers were collected
separately. Cells were washed three times with PBS and then treated with
trypsin for 5 min at 37°C to detach them from the filters and to remove
membrane-bound RBP. Cells were then transferred into plastic tubes, washed
three more times with PBS, and lysed with H2O on ice. The buffers
from the upper and lower chambers and the cell lysates were subjected to
precipitation with TCA. Radioactivity was measured with a counter both
in the precipitable and nonprecipitable material and in the trypsin wash.
ELISA and Western Blotting
For ELISA, 96-well microtiter plates were coated with the samples to be
tested for biotin-labeled proteins or unlabeled RBP. To detect biotin-labeled
proteins, we incubated wells with ALP-conjugated streptavidin (1:3000),
followed by p-Nitrophenyl-phosphate (Sigma). To detect unlabeled RBP, we
incubated wells with the anti-RBP antibody (1:500), followed by ALP-conjugated
anti-rabbit IgG (1:3000). Absorbance at 405 nM was determined with an E1-311
ELISA microplate reader. The amount of RBP was estimated using standard
curves, obtained by coating wells with 1 to 1000 ng of either unlabeled or
biotin-labeled RBP.
For Western blotting, samples to be tested for either unlabeled or biotin-labeled RBP or for other biotin-labeled proteins were subjected to 5 to 15% SDS-PAGE under nonreducing conditions and blotted onto nitrocellulose membranes, which were incubated with the rabbit anti-RBP antibody (1:200) followed by HRP-conjugated goat anti-rabbit IgG (1:2500) or, for biotin-labeled proteins, with HRP-conjugated streptavidin (1:500). Bands were detected using a chemiluminescence substrate kit (Kirkegard & Perry Laboratories, Gaithersburg, MD). The pixel density of the bands obtained by Western blotting was measured in scanned images using a personal computer software (NIH Imager 2.1).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RBP preparation used in the present study was purified from human urine, which is known to contain RBP complexed with retinol (23). To examine this further, we assessed the electrophoretic mobility of the RBP preparation under nondenaturing conditions. Under denaturing condition, retinol dissociates from RBP, with the result that RBP resolves into a single band. In contrast, under nondenaturing conditions, retinol remains complexed with RBP to some extent, resulting in slightly different electrophoretic mobilities of RBP not complexed with retinol (apo-RBP) and RBP complexed with retinol (holo-RBP) (17). As shown in Figure 1C, the RBP preparation used here resolved into two bands of similar intensities, indicating that it contained both apo- and holo-RBP in approximately similar amounts (approximately 47% apo- and approximately 53% holo-RBP, calculated by measurement of the pixel density of the bands).
We then tested the megalin-binding affinity of the RBP preparation, in solid phase assays. As shown in Figure 1D, 125I-labeled RBP bound to immobilized megalin in a concentration-dependent, saturable manner, and binding was inhibited by an excess of unlabeled RBP. The estimated constant of dissociation (Kd) was 834 nM. Thus, the affinity of the RBP preparation used here was similar to that reported in the study of Christensen et al. (6) (approximately 1000 nM).
Establishment of IRPT Cell Monolayers on Permeable Filters
To study transcytosis of RBP, we used IRPT cells cultured on permeable
filters in dual chambered devices. As described previously
(16), under these culture
conditions, IRPT cells are polarized, with megalin expression exclusively on
the upper facing surface. Furthermore, the cells form tight junctions, which
prevents appreciable paracellular leakage
(16).
To investigate the presence of tight junctions and paracellular leakage
with the IRPT cells used in the present experiments, we measured the TER of
the cell layers for several days after reaching confluence. As shown in
Figure 2A, there was a
progressive increase of TER values with a peak at day 9 (141.5 ± 37.2
x cm2), similar to that previously described
(16). In addition, we measured
transport across the cell layers of 3H-mannitol, a molecule of very
low mass (approximately 1 kD). After the addition of 3H-mannitol to
the upper chamber of 9-d confluent IRPT cells, the amount of
3H-mannitol transported in 6 h to the lower chamber was minimal
(1.40% of the amount added to the upper chamber), compared with the amount
transported through filters without cells (39.40%;
Figure 2B).
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RBP is Transcytosed across IRPT Cells
All transcytosis experiments were performed with 9-d confluent IRPT cells
on filters. RBP, either unlabeled or biotin-labeled, was added to the upper
surface of IRPT cells; after incubation at 37°C, fluids were collected
from the lower chamber and analyzed. As controls, we used biotin-labeled GST,
biotin-labeled BSA, or unlabeled lysozyme.
Both biotin-labeled RBP (Figure 3A, lane 2) and unlabeled RBP (Figure 3B) were found at approximately 21 kD by Western blotting in fluid collected from the lower chamber. In addition, when fluid from the lower chamber was subjected to immunoprecipitation with the anti-RBP antibody, Western blotting revealed a similar band (Figure 3A, lane 3), which provides further evidence that RBP was transported intact and with preservation of conformational epitopes.
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Because, as mentioned above, the RBP preparation used in the present study contained similar amounts of apo-RBP (not complexed with retinol) and holo-RBP (complexed with retinol), we investigated whether one of the two forms of RBP was transported preferentially across IRPT cells. For this purpose, we analyzed the material collected from the lower chamber by PAGE under nondenaturing conditions in transcytosis experiments with unlabeled RBP. As shown in Figure 3C, RBP in the lower chamber resolved into two bands of similar intensity, as did RBP added to the upper chamber (Figure 1C), indicating that apo- and holo-RBP were transported across IRPT cells to a similar extent. None of the control proteins was transported across IRPT cells. This was the case for GST and BSA (Figure 3D) and for lysozyme (Figure 3E), a protein of molecular mass slightly lower than that of RBP (approximately 15 kD).
By measuring the pixel density of the bands obtained by Western blotting, we estimated that approximately 6% of the RBP added to the upper chamber was transported to the lower chamber in 6 h. Approximately 70% of the RBP seen by Western blotting (Figure 3A, lane 2) was precipitated by the anti-RBP antibody (Figure 3A, lane 3).
As shown in Figure 3F, biotin-labeled RBP was also found in the lower chamber by ELISA, whereas biotin-labeled GST and BSA were not. The amount of RBP found in the lower chamber was markedly reduced when incubation was performed at 4°C, which supports the conclusion that RBP was transported by transcytosis and not by paracellular leakage. Based on ELISA results, we estimated that at 37°C, 17.5% of the amount of biotin-labeled RBP added to the upper chamber was transported to the lower chamber.
We then investigated simultaneously the transport of labeled RBP and
control substances across IRPT cells on filters. We first measured transport
of 125I-labeled RBP and 3H-mannitol. After incubation of
IRPT cells with 125I-labeled RBP and 3H-mannitol added
together to the upper chamber, radioactivity was measured in the buffer
collected from the lower chamber. 3H is known to emit only ß
radiation. However, 125I releases both and ß
radiation. To estimate the proportion of the total ß counts that resulted
from 125I-labeled RBP added to the upper chamber, we subtracted
ß counts produced by 125I-labeled RBP alone from total ß
counts produced by 125I-labeled RBP plus 3H-mannitol. We
determined that 125I-labeled RBP accounted for approximately 10% of
total ß counts. Therefore, approximately 90% of ß counts in samples
from the lower chamber were considered to represent 3H-mannitol
transported from the upper chamber. As shown in
Figure 4A, approximately 30% of
total counts added to the upper chamber were detected in the lower
chamber after 6 h of incubation, indicating that 125I-labeled RBP
had been transported to the lower chamber. The results are consistent with
those reported below, showing that after addition of 125I-labeled
RBP to the upper chamber of IRPT cells, approximately 15% transcytosed and
approximately 15% degraded RBP were found in the lower chamber. Only 3% of
ß counts added to the upper chamber were detected in the lower chamber,
indicating that transport of mannitol was considerably lower (approximately
10-fold) than transport of RBP, despite that RBP has a much higher
(approximately 20-fold) molecular mass. The results strongly support the
conclusion that the contribution of paracellular leakage to RBP transport
across IRPT cells is minimal.
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Similar experiments were performed to assess simultaneously the passage of biotin-labeled RBP and biotin-labeled lysozyme across IRPT cells. As shown in Figure 4B, after incubation of IRPT cells with both biotinylated proteins, only RBP transversed the monolayer, indicating that there was no measurable paracellular passage of lysozyme during RBP transcytosis. The results provide further evidence that paracellular leakage did not contribute appreciably to the transport of RBP across IRPT cells.
We also studied transport of RBP by immunofluorescence staining with confocal microscopy, after addition of unlabeled RBP to the upper chamber of IRPT cells on filters. RBP was revealed by the anti-RBP antibody. As shown in Figure 5, punctate staining was seen after 10 min of incubation near or on the apical membrane (possibly associated with clathrin coated pits; Figure 5A), as well as at deeper levels in the cells (Figure 5, B through D), where staining was predominantly seen in the peripheral region of the cells, consistent with the interpretation that vesicles that contain RBP were close to or associated with lateral membranes. A similar pattern was seen in cells that were incubated with RBP for 60 min (not shown), which indicates that the lesser intensity of staining seen in the deeper levels of the cells was not entirely due to insufficient time for the endocytosed RBP to reach the basal portion of the cells. Instead, the results may be explained by degradation of some RBP in lysosomes, as well as by release of RBP at lateral membranes, below the level of tight junctions. No staining was seen in cells that were incubated with buffer that lacked RBP (not shown). This finding demonstrates that the anti-RBP antibody did not recognize proteins other than exogenous RBP in IRPT cells and that the staining seen in cells that were incubated with exogenous RBP was specific. Overall, although the confocal microscopy experiments by themselves do not provide direct evidence for transcytosis, they are consistent with the conclusions supported by the other experiments described above, which show that some RBP is transcytosed across IRPT cells and in particular suggest that some RBP exits the cells from lateral cell membranes, below the level of tight junctions.
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Transcytosis of RBP Results from Megalin-Mediated Endocytosis
To investigate whether and to what extent transcytosis of RBP occurs as a
consequence of megalin-mediated endocytosis by IRPT cells, we performed
experiments with megalin competitors. As shown in
Figure 6A, when unlabeled RBP
was added to IRPT cells together with the megalin competitors, namely RAP-GST
or the anti-megalin antibody 1H2, passage of RBP from the upper to the lower
chamber was reduced, as assessed by Western blotting. By measuring the pixel
density of the RBP band, we estimated that the extent of inhibition produced
was approximately 75% by either RAP-GST or 1H2, whereas no appreciable effects
were seen after co-incubation with GST or normal mouse IgG, used as
controls.
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Similar results were obtained when biotin-labeled RBP was used, and the amount transcytosed was measured by ELISA. As shown in Figure 6B, when biotin-labeled RBP was added to IRPT cells together with RAP-GST or 1H2, passage was reduced by approximately 90%, whereas no effect was produced by GST or normal mouse IgG.
Lysosomal Degradation of RBP Is Not Affected by Megalin
Competitors
Cells that express megalin are known to degrade some exogenously added RBP,
as previously reported (6).
However, it has not been determined to what extent this is a consequence of
megalin-mediated endocytosis. To answer this question, we performed
experiments to determine degradation of 125I-labeled RBP by IRPT
cells (cultured on plastic), as detailed in the Materials and Methods section.
As shown in Figure 7,
approximately 18% of the amount of 125I-labeled RBP added was
degraded by IRPT cells. RBP degradation was reduced by approximately 40% after
pretreatment of the cells with chloroquine. However, when
125I-labeled RBP was added to the cells in the presence of RAP-GST
or 1H2, no significant reduction of degradation was seen
(Figure 7), indicating that
little, if any, of the degraded RBP had been internalized by megalin. The
results suggest that under the conditions studied, mechanisms of uptake other
than megalin-mediated endocytosis account for RBP transport to lysosomes.
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To calculate the proportions of RBP internalized by IRPT cells that underwent transcytosis or degradation, we performed experiments with IRPT cells on filters in which transcytosis and degradation of RBP were measured simultaneously. After incubation of IRPT cells with 125I-labeled RBP added to the upper chamber, cells were treated with trypsin and lysed. The buffers from the upper and lower chambers and the cell lysates were subjected to precipitation with TCA. As shown in Figure 8A, the proportions of the total amount of 125I-labeled RBP added to IRPT cells were found to be distributed as follows: (1) upper chamber: 3.4% non-TCA precipitable (degraded) and 33.3% TCA precipitable (unprocessed or recycled); (2) lower chamber: 14.3% non-TCA precipitable (degraded) and 14.9% TCA precipitable (transcytosed); (3) cell lysate: 1.5% non-TCA precipitable (degraded) and 0.29% TCA precipitable (internalized and undegraded); and (4) trypsin wash: 30.0% (cell bound). On the basis of these results, we estimated the proportions of total internalized RBP that were either degraded or transcytosed. Total internalized RBP comprised the following fractions: (1) transcytosed RBP (TCA-precipitable material in the lower chamber), (2) degraded RBP (non—TCA-precipitable material from both the lower and the upper chambers and from the cell lysate), and (3) intracellular RBP that had not been degraded (TCA-precipitable material in the cell lysate). As shown in Figure 8B, the proportions of total internalized RBP were distributed as follows: 0.78% (0.29% of total RBP added) intracellular undegraded, 43.2% (14.9% of total RBP added) transcytosed, and 55.8% (19.2% of total RBP added) degraded.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several considerations support the conclusion that passage of RBP from the upper to the lower chamber was by transcytosis rather than by paracellular leakage: (1) passage was markedly reduced in experiments performed at 4°C, at which temperature transcytosis is abrogated (22); (2) passage was reduced by megalin competitors, which provides evidence that the process was specific, as expected of receptor-mediated transcytosis but not of paracellular leakage; and (3) as reported previously in a study of transcytosis of thyroglobulin (16), IRPT cells cultured under the conditions used here form a tight junctional barrier, as shown by the presence of intercellular junctional complexes close to the apical surface detected by electron microscopy and by the expression of the tight junction-associated protein occludin (24). Evidence that the cells used in the present experiments had tight junctions was shown by a progressive increase of TER during several days of culture and by the relatively low passage of 3H-mannitol across cell layers, a molecule of small mass (1 kD). In addition, we provided evidence here that there was no appreciable paracellular leakage across IRPT cells of two biotin-labeled proteins, namely GST and BSA, or of another megalin ligand, namely lysozyme, which has a molecular mass slightly lower than RBP.
Our conclusion that the direction of RBP transcytosis was from the apical to the basolateral cell surface is based on evidence obtained in a previous study (16), which showed that when IRPT cells are cultured under the conditions used here, they exhibit polarity, with the apical surface facing the upper chamber. Thus, megalin—an apical membrane protein (9,10)—is expressed by these cells exclusively on the upper surface of the cell layer (16). In addition, electron microscopy revealed microvilli and coated pits exclusively on the upper surface of the cell monolayer (16). In the present study, we showed by confocal microscopy that at 10 and 60 min after addition of RBP to the apical surface of polarized IRPT cells, RBP was concentrated in vesicles at the upper surface but was also present at deeper levels of the cell, where numerous vesicles appeared to be close to basolateral membranes. Because of their predominantly peripheral location near or at the cell membranes, it is unlikely that many of the RBP-containing granules represented lysosomes, although, as shown in the present study, some RBP internalized by IRPT cells undergoes lysosomal degradation. However, we obtained evidence that megalin-mediated endocytosis did not result in appreciable lysosomal degradation of RBP. Thus, megalin competitors failed to reduce the extent of RBP degradation. This suggests that other mechanisms of RBP uptake—either other receptors or fluid phase pinocytosis—may be responsible for RBP lysosomal degradation in IRPT cells. However, further studies are needed to determine the importance of these mechanisms. We estimated that 45% of the RBP taken up by IRPT cells underwent transcytosis and approximately 55% underwent lysosomal degradation. However, if RBP uptake by renal proximal tubule cells in vivo is mediated almost entirely by megalin—as suggested by the absence of RBP-containing granules in the proximal tubule of megalin knockout mice (6)—then the proportion of RBP internalized from the glomerular filtrate and transcytosed across renal proximal tubule cells should be considerably higher.
Transcytosis provides a plausible explanation for the way in which vitamin A (retinol) that is filtered through the glomerulus bound to RBP is returned to the circulation. As noted earlier, an alternative pathway was proposed by Christensen et al. (6), namely that after megalin-mediated endocytosis, RBP-retinol is transported to lysosomes, where retinol is released and subsequently combines with newly synthesized RBP, after which RBP-retinol is secreted into the circulation. This interpretation depends on the assumption that RBP is synthesized in the same cells in which it is absorbed. However, Makover et al. (25) reported that RBP mRNA is located in sites that differ from RBP protein in rat kidneys: mRNA is found in the outer stripe of the medulla, mainly in the S3 segment of proximal tubules, whereas RBP protein is seen predominantly in the early segments (6,25). Furthermore, as reported by Christensen et al. (6), in megalin knockout mice, there was no staining for RBP in proximal tubule cells, which indicates that the RBP seen in wild-type mice represented absorbed rather than endogenously synthesized RBP, a conclusion supported further by the pattern of immunohistochemical staining for RBP, namely in coarse granules, which are not characteristic of proteins in the endoplasmic reticulum. Although much of the granular staining for RBP in proximal tubules of normal mice, rats, or humans illustrated in the report of Christensen et al. (6) is subapical, some granules are seen clearly at deeper levels, including some near the basal surface, especially in human and rat kidneys. There is no evidence that RBP can be absorbed at the basal surface from the circulation, as occurs with some other proteins (26). Thus, some of the RBP-containing granules seen in proximal tubule cells in kidney sections (6), especially those in deeper levels of the cells, may be RBP-containing vesicles undergoing transcytosis. A somewhat different pattern of staining in proximal tubule cells in normal kidneys is seen with lysozyme, another megalin ligand that is present in low concentrations in the serum and that is reabsorbed from the glomerular filtrate (27). Thus, lysozyme is found almost exclusively in subapical granules, as illustrated in a report from Leheste et al. (27). As we show in the present study, lysozyme added to the upper surface of cultured IRPT cells did not transverse the cell layer, showing that it did not undergo transcytosis. It is therefore likely that lysozyme, like most ligands endocytosed by megalin, is transported to lysosomes and degraded.
Findings obtained in a different type of study by Gjoen et al. (28) provide indirect evidence of transcellular passage of intact RBP after its cellular uptake in vivo and support our conclusion that RBP is transcytosed across renal proximal tubule cells. These investigators used the "trapped ligand technique" to study catabolism of RBP (28). The method makes use of a nonmetabolizable tracer, such as 125I-tyramine cellobiose (TC), which is linked covalently to the protein being studied. After tissue uptake and degradation of the protein, 125I-TC is retained in the cells, where it serves as a marker of degradation of the protein (29). Gjoen et al. (28) prepared 125I-TC—labeled rat RBP and studied its distribution after intravenous injection into rats. Between 5 to 24 h after injection, approximately 30% of the radioactivity originally taken up in the liver and kidney was lost. The authors postulated that the loss of radioactivity indicated that some of the labeled RBP escaped degradation and was released from cells, possibly by a process they called "retroendocytosis," which in the light of present knowledge could include transcytosis as well as recycling of the ligand to the cell surface at which it was endocytosed.
Evidence indicates that certain proteins are transported intact from the lumen of the proximal tubule to the peritubular space, but the extent to which this results from transcytosis rather than paracellular leakage is controversial (reviewed in reference 26). Certain microperfusion studies that purported to show transcytosis have been criticized because increased intraluminal pressure may have disrupted junctional complexes, leading to paracellular leakage (26). Furthermore, electron microscopic studies of peroxidase transport by renal proximal tubule cells have revealed peroxidase in intercellular spaces, which was considered evidence of paracellular leakage (30). However, apical to basolateral transcytosis can take place by release of ligands at the lateral membrane into the intercellular space anywhere below the level of tight junctions, as has been shown to occur during absorption of IgG across the intestinal epithelium of neonatal rats (31). In this regard, junctional complexes of proximal tubule cells (including IRPT cells) are fairly close to the apical surface. Indeed, findings made by confocal microscopy in the present study provide indirect support for release of RBP-containing vesicles at lateral membranes.
A number of previous studies of protein handling by cultured renal cells have been criticized, in large part because the cells used either were not derived from proximal tubules or, if they were, as in primary cultures, did not clearly resemble proximal tubule cells in vivo (26). However, the IRPT cells used here are clearly of proximal tubule origin and exhibit many of the features of the cells seen in vivo, including apical megalin expression, brush borders, and tight junctions (16). Furthermore, we have shown that megalin, the major endocytic receptor on proximal tubule cells (9,10,11,12), is functional in IRPT cells (16,20).
As noted earlier, most ligands internalized by megalin are transported via endocytic vesicles to lysosomes, resulting in dissociation and degradation of ligands and recycling of the receptor, as has been shown in various cultured cells that express megalin (11,12,13,14,15). However, we recently obtained evidence, both with cultured cells and in vivo, that binding and uptake of thyroglobulin via megalin on thyroid cells results in apical to basolateral transcytosis rather than in delivery to lysosomes (16). In that study (16), we also found that transcytosis of thyroglobulin via megalin occurs not only in thyroid cells but also in IRPT cells. Furthermore, we showed that another megalin ligand, lactoferrin, was degraded but not transcytosed after internalization via megalin both in thyroid and IRPT cells, suggesting that transcytosis via megalin across these cells depends on the ligand (16). However, in certain cell types, megalin may be responsible for transcytosis of ligands that are known to be transported to lysosomes in other megalin-expressing cells. For example, apolipoprotein J (clusterin) can be transported across cerebral endothelial cells via megalin, whereas this ligand is degraded in other cells (32,33). Moreover, several other receptors, including members of the LDL receptor family, can mediate transcytosis of certain ligands selectively across cerebral endothelial cells, suggesting that those cells have special mechanisms that divert endocytosed ligands to the transcytotic pathway. For example, LRP-mediated endocytosis of lactoferrin results in transcytosis across the blood-brain barrier, but it leads to degradation of the ligand in epithelial cells (34). The mechanisms that lead to lysosomal transport or (less common) to transcytosis of ligands internalized by megalin are not known and require further investigation.
Our present findings extend the physiologic role of megalin in transcytosis beyond the thyroid (16) and the blood-brain barrier (32). It may be that transcytosis accounts for recycling of certain megalin ligands other than RBP that have been filtered through the glomerulus (27). In addition, it is possible that megalin-mediated endocytosis results in transcytosis of certain ligands across other epithelial cells. Further studies are needed to investigate these possibilities.
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