Megalin binds and internalizes angiotensin II - 《American Journal of Physiology》美国生理学杂志干细胞之家

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发表于 2009-4-21 13:06 |只看该作者 |倒序浏览 |打印

Tulane Hypertension and Renal Center of Excellence and Department of Physiology, Tulane University School of Medicine

Department of Chemistry, Xavier University of Louisiana" M( Q* H8 B/ p! x) v$ W' D0 p

Department of Medicine/Section of Nephrology, Tulane University School of Medicine, Tulane Veterans Affairs Environmental Astrobiology Center

Veterans Affairs Medical Center, New Orleans, Louisiana, a# k& S# Y' i6 c+ D9 _# ?1 \. x
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ABSTRACT

Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels.

endocytosis; epithelial cells; angiotensin type 1 receptor; intracellular angiotensin II9 y! \$ v( j: ~: Y* F9 D

ANGIOTENSIN II (ANG II), the main effector of the renin-angiotensin system (RAS), exerts powerful effects on epithelia from organs as diverse as the placenta (37) and the kidney (34). In this sense, the proximal tubule (PT) has served as the prototype for ANG II studies in epithelial cells and therefore has been extensively studied. In the PT, ANG II stimulates several physiological processes such as sodium and water reabsorption (12), luminal acidification (31), several hormonal systems like the RAS (22, 23, 27), and several nuclear factors (7). Furthermore, ANG II has been linked to several seemingly interrelated pathophysiological processes that ultimately lead to kidney damage, including generation of reactive oxygen species (18), cell hypertrophy (19, 49), the production of extracellular matrix proteins (50), and/or apoptosis (6).

Epithelial cells targeted by ANG II, such as PT and placental epithelial cells, generally express both types of angiotensin receptors (AT1R and AT2R) (13). The effects of ANG II however, are mediated predominantly by ligand binding to AT1R, which is expressed at both the apical and basolateral surfaces of the PT (9, 10). Furthermore, ANG II is internalized on binding to AT1R and this process has been linked to the regulation of NaCl transport and to the activation of distinct intracellular pathways at the apical surface of PT cells (2, 4, 5, 40, 41). Importantly, ANG II accumulation in the kidney, via AT1R internalization, seems to play a role in the regulation of transport and the pathogenesis of several forms of ANG II-dependent hypertension (35). Furthermore, both ANG II infusion and low-salt diet increase tissue ANG II concentrations in the kidney and this effect seems to be AT1R mediated, as demonstrated by its inhibition through the use of AT1R blockers (ARBs) (24, 53).7 m6 g/ J* e2 ^6 i5 [, H

We initiated a series of experiments to investigate the AT1R internalization-dependent and -independent effects of ANG II. Several lines of evidence derived from transfection of truncated AT1R constructs, site-directed mutagenesis, and site-specific peptide inhibitors have implicated the Thr332, Ser335, Thr336, Leu337, and Ser338 motif in the cytoplasmic tail of the receptor as one of the predominant sites mediating AT1R internalization (21, 42, 44). In the course of studies on mechanisms of cellular ANG II uptake, it was found that permeable peptides matching this putative internalization sequence only partially inhibited AT1R internalization in an ANG II uptake study in placental epithelial cells. Furthermore, when candesartan (an ARB) was studied in the same assay as a positive control for AT1R inhibition of ANG II binding, we observed a similar result with again only partial inhibition of ANG II uptake. These findings suggested not only that there was another internalization motif in the intracellular portions of AT1R, but also that there was an alternate uptake pathway for ANG II.
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Of the reabsorptive mechanisms known to be active at the apical surface of the placental epithelial cell, the megalin/cubilin complex seemed to us as the best candidate to explore as an alternate ANG II uptake pathway as it is appropriately distributed and sufficiently abundant to play a role in ANG II uptake (47). Accordingly, our experimental studies were designed to evaluate the role of the megalin/cubilin complex as a receptor for ANG II.
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MATERIALS AND METHODS% @9 i% H, [& P# C9 X% W- S, o5 w7 j" A" o
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Animals, Reagents, and Antibodies6 h& n+ Z1 `0 v% ~  B; b! K

Mice (CD-1 strain, 6–12 mo old, n = 30) were purchased from Charles River Laboratories (Wilmington, MA). These animals were kept in a temperature-controlled room and fed standard Purina mouse chow diet with free access to tap water. All reagents were from Sigma (St. Louis, MO) unless otherwise stated. Unlabeled ANG II (U-ANG II) was purchased from Phoenix Pharmaceuticals (Belmont, CA). Alexa Fluor 488-conjugated ANG II (Alexa 488-ANG II) was obtained from Molecular Probes (Eugene, OR). Purified human megalin and polyclonal antibodies against cubilin and megalin were provided by Dr. P. J. Verroust (Institut National de la Santé et de la Recherche Médicale, Paris, France). These antibodies were raised against proteins purified by immunoaffinity chromatography using previously reported monoclonal antibodies coupled to Sepharose 4B (17, 32, 38, 39). Antibodies were determined to be monospecific by immunoblotting on whole brush-border preparations and by immunoprecipitation of biosynthetically labeled yolk sac epithelial cells in culture. Importantly, these antibodies recognize mouse, human, and rat megalin (38, 39). Anti-neurokinin-1/substance P receptor antiserum (anti-NK1 antibodies) was a gift of Dr. J. Couraud, Gif-sur-Yvette, France (8). A rabbit polyclonal antibody against an epitope near the NH2 terminus of AT1R (N-10) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Candesartan was provided by AstraZeneca (Sweden). Custom peptides were purchased from Biosource (Worcester, MA). Custom oligonucleotides were purchased from Qiagen (Alameda, CA). TRIzol LS Reagent, SuperScript One-Step RT-PCR with Platinum Taq kit, and 1 kb PLUS DNA Ladder were obtained from Invitrogen Life Technologies (Carlsbad, CA).: M7 D2 n8 H0 `; x3 V

Cell Culture4 k  ]! e9 Y+ i% D* W( C: U
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Cell experiments were conducted using immortalized yolk sac cells from the Brown Norway rat (BN-16 cells) (passages 1-12) (29). These cells possess several characteristics that resemble PT cells and make them suitable for endocytosis studies including a well-developed brush border and a specialized endosomal pathway similar to the renal PT, with abundant expression of megalin, cubilin, and surface epithelial markers specific for the brush border (29). BN-16 cells were cultured in conventional T-flasks (T225 or T75) in a humidified chamber with 5% CO2 and environmental air using DMEM/F-12 (Gibco/Invitrogen, Carlsbad, CA) supplemented with heat-inactivated fetal calf serum (10%) and an antibiotic cocktail Ciprofloxacin (Bayer, West Haven, CT) and Fungizone (Gibco/Invitrogen). Cells were fed every 48 h and divided on reaching confluency.

AT1R Expression Studies in BN-16 Cells/ \- d  Q' o, |0 H8 Z3 w. b
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RNA isolation and amplification by RT-PCR of AT1aR and AT1bR loci. Total RNA was isolated from BN-16 cells cultured in monolayers using the TRIzol LS Reagent method; manufacturer's recommendations were followed. RNA was frozen at –80°C until used. The sequences for oligonucleotide primers specific for each subtype of AT1R receptor used in this work have been previously described and tested for subtype specificity (20). RT-PCR amplification was performed using the SuperScript One-Step RT-PCR with Platinum Taq kit from Life Technologies. The thermocycling protocol was as follows: reverse transcription 50°C 30 min, 94°C 1 min. PCR amplification (28 cycles) included 94°C 30 s, 58°C 60 s, 72°C 60 s, and 72°C 5 min. RT-PCR products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, visualized by UV transillumination, and documented using a Kodak Digital Science Electrophoresis Documentation and Analysis System (Eastman Kodak Rochester, NY).7 D8 Y* I2 e. u2 J
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Surface expression of AT1R protein by antibody binding techniques. The expression of the AT1R protein at the cell surface was determined using membrane vesicles (MV) isolated from BN-16 cells growing in conventional T75 flasks. The protocol followed to extract MV from cells in culture has been described elsewhere (25). MV in suspension in PBS solution were preincubated with goat serum to avoid nonspecific binding. MV were then spun down, washed, and resuspended in PBS containing a rabbit polyclonal antibody against the NH2 terminus of AT1R (in serial dilutions 1:50–1:20,000). After 1-h incubation at 37°C, a second wash, and resuspension, MV were treated with a R-phycoerythrin (FITC)-conjugated goat anti-rabbit antibody against rabbit IgG (diluted to 1:80 following the manufacturer's recommendations). Samples were analyzed using a FACS Vantage flow cytometer (Becton Dickinson Immunocytochemistry, San Jose, CA) using a dedicated Mac computer. Excitation was at 488 nm using a coherent 6-W argon ion laser. For each particle, emission was measured using photomultipliers at 530 ± 30 nm. Data were collected as 2,000 observations-mode files and were analyzed using CellQuest software (Becton Dickinson Immunocytochemistry).6 p/ y' W# P; F  \9 A$ w
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ANG II Uptake in BN-16 Cells Analyzed by Flow Cytometry

Uptake experiments were performed using BN-16 cells grown in monolayers and exposed to labeled ANG II [Alexa 488-ANG II (100 nM)] for the indicated times with or without a competitive antagonist.
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Effect of Myristoylated Peptides and Candesartan

For AT1R-dependent ANG II uptake inhibition experiments, two different approaches were used: in the first one, a myristoylated peptide matching an internalization motif in the cytoplasmic tail was used to prevent the internalization without affecting binding. The sequence of the peptide (Myr-LSTKMSTLSY-OH) (Inhibitor Peptide or IP) matches a sequence (Thr332, Ser335, Thr336, Leu337, and Ser338) in the cytoplasmic tail of the AT1R involved in the internalization of this receptor (21, 42). This peptide has been used to prevent membrane fusion in vitro (3). A separate peptide with the reverse sequence was used as a control (Myr-RYSLTSMKTSL-OH) (Control Peptide or CP). Additionally, chloroquine (1 mM) was used as a negative control because it prevents uptake and/or accumulation via the endocytic/lysosomal pathway (14). In the second approach, candesartan was used at a concentration 1,000-fold greater than ANG II (10 μM) to prevent binding of ANG II to the AT1R.' J! U- s7 F5 D7 o* ~! Q
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BN-16 cells were seeded in 96-well plates (20,000 cells/well) and allowed to grow to confluence. Next, cells were washed with serum-free DMEM (SFM) and allowed to equilibrate for 2 h at 37°C. Candesartan or the peptides (10 μM each) in SFM were added 30 min before Alexa 488-ANG II. Then, Alexa 488-ANG II in SFM was added to a final concentration of 100 nM. Subsequently, the cells were incubated at 37°C and the internalization process was stopped at the indicated times (see RESULTS). To stop the reaction and remove unbound Alexa 488-ANG II, the well plate was placed on ice and the cells were washed twice with ice-cold PBS. The cells were incubated for 5 min with an acid solution to release membrane-bound Alexa 488-ANG II. At this stage, cells were washed again with PBS and trypsinized, transferred to flow cytometry tubes, and stored on ice briefly until the cell-associated fluorescence (an indication of internalized Alexa 488-ANG II) was analyzed by flow cytometry as previously described.  i( v+ L9 @; ^6 n, x
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Effect of Anti-megalin and Anti-cubilin Sera# B* R! W( V# Q) T) t
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The uptake of Alexa 488-ANG II by BN-16 cells was analyzed in the presence of 100- to 3,000-fold dilutions of polyclonal antibodies anti-megalin and anti-cubilin that recognize the holoprotein (1, 17, 38, 39). In a previous study these antibodies were shown to effectively prevent the uptake of known ligands for megalin and/or cubilin (26). Anti-NK1 peptide antibodies were chosen as negative controls for nonspecific interference by binding because they bind brush-border MV at the same titer as the anti-megalin antisera (26). Other additional experimental groups were included for further analysis: candesartan was added to a combined megalin/cubilin antisera to search for an additive effect; candesartan was used in a separate group as a means of comparison with ANG II uptake via AT1R; and chloroquine was used to demonstrate that Alexa 488-ANG II uptake is endocytotic in nature.* g/ A4 _3 G: [8 u: |

These experiments were performed using the same protocol, with the following modifications: to prevent nonspecific binding, ovalbumin (0.1%) was added to the SFM and this solution (SFM-ovalbumin 0.1%) was used at all steps from equilibration. The antibodies anti-megalin, anti-cubilin, as well as a combination of both and anti-NK1 were added after equilibration to the appropriate wells and then the cells were incubated for 1 h at 37°C before adding Alexa 488-ANG II. After the preincubation step, Alexa 488-ANG II was added to a final concentration of 100 nM and the reaction stopped after 4 h.
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Preparation of Fluorophore-Conjugated Metallothionein" N; J9 R! ^" W+ T

For those experiments using labeled metallothionein (MT), the protein was conjugated to cyanine (Cy3) (Molecular Probes) following the supplier's protocols. Because MT is a very small protein, unreacted dye was removed by dialysis against PBS at pH 7.4 in Slide-A-Lyzer dialysis cassettes having 3,500-kDa molecular mass cut off (cat. no. 66330, Pierce, Rockford, IL) rather than using the columns provided in the manufacturer's kit.

Effect of ANG II in the Uptake of Cy3-MT
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Because previous studies demonstrated that MT is a ligand for megalin (26), Cy3-labeled MT (20 μM) was chosen for competition experiments with ANG II. Competition experiments were performed according to the general protocol with the following modifications: cells were preincubated with increasing concentrations of U-ANG II. After 4 h, 20 μM Cy3-MT was added and the cells were incubated for an additional hour at 37°C. Positive controls were not preincubated with ANG II. Cell-associated fluorescence was analyzed by flow cytometry collecting the cyanine-3 signal at 575 ± 26 nm.8 J* Q% H' P/ o& l) m% m& X2 A. _7 p
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ANG II Binding to Brush-Border MV Analyzed by Flow Cytometry
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On the day of the experiment, mice were killed by CO2 inhalation in a closed chamber. The kidneys were then extracted via median incision, randomly distributed in four groups (15 kidneys per group), and kept in ice-cold PBS until further processing. Next, brush-border MV (BBMV) were isolated according to the divalent cation precipitation method (1, 17, 32). The binding of ANG II to BBMV was investigated in the presence of competitors for AT1R binding (candesartan 10 μM) or megalin/cubilin binding (anti-cubilin antibodies, anti-megalin antibodies, and combination of anti-megalin and anti-cubilin antibodies 1/100 dilution each and MT 20 μM). BBMV were preincubated for an hour with SFM-ovalbumin 0.1% at 4°C for an hour and then the different competitors were added and the BBMV were incubated for another hour at 37°C before Alexa 488-ANG II was added. Finally, BBMV were incubated for 4 more h at 37°C and the binding of Alexa 488-ANG II to BBMV was analyzed by flow cytometry as described.- w% E* K) ?( |1 Y

Surface Plasmon Resonance Experiments! p" L8 y7 q  q

The interaction of ANG II with megalin was assayed using a BIACORE 3000 biosensor system (Biacore AB). For these experiments, megalin was immobilized to a dextran-coated gold surface (CM5 biosensor chip). In surface plasmon resonance (SPR), injection of a soluble protein produces a signal change that is directly proportional to the mass of bound protein and is reported as resonance units (RU). Thus SPR allowed us to explore qualitatively "an interaction" between megalin and ANG II. Megalin (0.025 mg/ml in 10 mM acetate, pH 4.53) was immobilized (6,000–8,000 RU) in one flow cell on a CM5 chip using standard primary amine-coupling methods as detailed by the manufacturer. A second flow cell was activated and blocked with ethanolamine but lacking immobilized megalin, providing a real-time reference correction for instrumental artifacts and nonspecific binding events. ANG II (0.5 mg/ml) was injected over both flow cells at room temperature in HEPES-buffered saline (HBS), pH 7.4, containing 2 mM Ca, 2 mM Mg, and 0.005% surfactant P20. Maximum reproducibility was obtained when 0.0008% sodium dextran sulfate (Pharmacia Biotech, cat. no. 17–0340-01) was also included in the buffer. ANG II was injected at a flow of 5 μl/min for 25 min and then allowed to dissociate for 15 min. The small size of ANG II (1 kDa) enabled us to use a slow flow rate without encountering problems associated with diffusion (mass transport). Because we found ANG II to be a low-affinity ligand, no regeneration (removal of bound protein by injection of a second, typically harsh, solvent) was necessary. The "double-referencing" technique of Myszka (33) was used to eliminate additional instrumental artifacts. The blank injections used for this procedure were identical to sample solutions except for the omission of ANG II.

The receptor cubilin was immobilized under virtually identical conditions. Cubilin (0.025 mg/ml in 10 mM acetate, pH 4.78) was immobilized (6,000–8,000 RU) in one flow cell on a CM5 chip using standard primary amine-coupling methods as detailed by the manufacturer. A second flow cell was activated and blocked with ethanolamine but lacked immobilized cubilin, providing a real-time reference correction for instrumental artifacts and nonspecific binding events. ANG II (0.5 mg/ml) was injected over both flow cells at room temperature in HBS, pH 7.4, containing 2 mM Ca, 2 mM Mg, and 0.005% surfactant P20. ANG II was injected at a rapid flow of 50 μl/min for 2.5 min and then allowed to dissociate for 10 min. Because no binding was observed, regeneration (removal of bound protein by injection of a second, typically harsh, solvent) was unnecessary. Additional procedures were as described above for megalin.- o9 W/ O; q; O; l* C( E0 `1 I

Statistical Analysis
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Data are expressed as means ± SD or means ± SE throughout the manuscript. Statistical analysis was performed using Graph Pad Prism Software 3.0 (Graph Pad Software) by one-way ANOVA (and Tukey's post hoc comparison) and/or two-way ANOVA (and Bonferroni's post hoc comparison) whenever they were necessary. Flow cytometry data were also analyzed by Kolgomorov-Smirnov summation statistics (52). A value of P , T3 D' j/ Q0 E3 k
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RESULTS
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Expression of AT1R in BN-16 Cells0 m+ z9 \" a' K: @- J' Z/ p
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The presence of AT1R in BN-16 cells was tested at both the gene and protein level. First, RT-PCR using the specific primers for AT1aR and for AT1bR loci was followed by gel electrophoresis separation of products. mRNA expression of both genes was confirmed (Fig. 1A). Next, the expression of the protein at the cell surface was investigated using MV exposed to a serial dilution of a primary antibody targeted against an epitope in the NH2 terminus of AT1R. A secondary FITC-conjugated antibody was used to determine primary antibody AT1R binding by flow cytometry. The presence of AT1R was determined by the observation of a specific binding peak at a dilution of 1/5,000 of the primary antibody (Fig. 1B).
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Alexa 488-ANG II Uptake of BN-16 Cells Determined by Flow Cytometry
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Figure 2 shows that BN-16 cells avidly took up ANG II and the process seems to reach a plateau at 24 h (data after 24 h not shown).4 [; x$ L4 m  D+ u0 z

Role of AT1R in ANG II Uptake by BN-16 Cells: Effect of the Peptides (IP and CP) and Candesartan
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In tests for ANG II uptake via AT1R inhibition by the permeable peptides (Fig. 2A), the cumulative effect of the different agents (IP, CP, and chloroquine) accounted for 37.49% of variance (P

ANG II uptake by BN-16 cells in this series of experiments was a time-dependent process (time accounted for 36.42% of total variance P 0 J% ?$ s$ k" s" P
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In tests for effect of candesartan (Fig. 2B), the action of this agent (ANG II vs. ANG II candesartan) accounted only for 1.2% of the general variance (2-way ANOVA, P & Q5 {* f- v! Y2 C6 k
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Importantly, as was the case for the IP, candesartan did not prevent completely at any stage the accumulation of cell-associated fluorescence (i.e., ANG II internalization). Thus none of the maneuvers implemented to block ANG II uptake via AT1R completely abolished this process.: {  I" _9 y# {  T' `3 i

Effect of Anti-Megalin and Anti-Cubilin Sera
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We tested our hypothesis (megalin-mediated ANG II uptake) by using confluent monolayers of BN-16 cells preincubated with interfering polyclonal sera raised against megalin, cubilin, or a combination of both. Figure 3A shows the results of such experiment after 4-h incubation. We chose this time point based on practical considerations as well as on the previous experiments where enough ANG II accumulation was seen while only partial inhibition of ANG II uptake via AT1R was obtained.* s) ]2 i6 O1 [3 B% \  I3 F. T& {

Considered separately, anti-megalin antibodies produced the strongest inhibition of ANG II uptake of all antisera (P 9 A6 _* S3 Y3 b5 G; v
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Candesartan also produced a small but significant effect compared with positive control. This effect was lower than the one produced by the lowest dilutions of the anti-megalin antibody (P

Cy3-MT Uptake Via Megalin in BN-16 Cells: Competition Experiments with U-ANG II Analyzed by Flow Cytometry# C$ n7 {: c. Z  d6 o0 S. F
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The rationale of this experiment was to demonstrate competition for uptake between a known ligand (MT) and a potential ligand (ANG II) for megalin. For this, BN-16 cells were preincubated with ANG II for 4 h and then coincubated with labeled MT (Cy3-MT) and ANG II (Fig. 3B). After 4-h incubation, ANG II interfered with MT uptake (P
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ANG II Binding to BBMV Analyzed by Flow Cytometry
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Figure 4, top, shows the results of experiments aimed to analyze Alexa 488-ANG II binding to BBMV. A general variability was detected by ANOVA (P # o9 z& m* T/ F
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Molecular Studies of ANG II-Megalin Binding Using SPR$ O$ k& A' `, v) X1 s& Z

A sample of the analysis by SPR of the ANG II binding to megalin is shown in Fig. 4, middle. Surface mass changes compatible with a low-affinity interaction between megalin and ANG II were detected. Moreover, these responses uniformly increased with time. The observed variations and noise are normal for the very low signal levels used to optimize a study of binding constants when the sizes of the two interacting species are quite different. To produce signal changes large enough for qualitative detection of binding, we were compelled to use high surface coverage by megalin on the SPR chip. This enabled us to observe reproducible evidence of binding, but at the cost of obtaining the kinetic parameters associated with the binding.5 z) m1 U, u" w
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During the dissociation of ANG II from megalin, the signal decayed exponentially with a half-life of 15 s, consistent with a low-affinity binding interaction; in contrast, the signal recorded for the interaction between ANG II and cubilin remained at the baseline during both association and dissociation phases, indicating that no binding occurred (Fig. 4, bottom).
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DISCUSSION

The results show by two different approaches that inhibition of ANG II uptake into BN-16 cells via AT1R only partly reduces ANG II internalization, at least in the first 24 h of ANG II exposure. We then present four lines of evidence supporting our hypothesis that megalin is a receptor for ANG II. First, BN-16 cells were exposed to several antisera specific for megalin, cubilin, or a combination of both. Among the different groups, anti-megalin antibodies inhibited ANG II uptake the most strongly. In the second line of evidence, uptake in BN-16 cells of a known ligand for megalin (Cy3-MT) was significantly attenuated by preincubation and coincubation with pure ANG II. The third line of evidence is represented by ANG II binding experiments to BBMV. In these experiments, the use of either anti-megalin antibodies alone or a combination of anti-megalin/anti-cubilin antibodies reduced significantly the binding of ANG II to BBMV. Thus, in a freshly isolated animal preparation representative of the apical membrane of the PT, the data support the role of megalin as a receptor for ANG II. Last, SPR experiments and analysis are consistent with direct binding of ANG II to purified megalin. An injection of ANG II produced a small change in mass at the surface of megalin receptor-coated chip. This change of mass, although quite small, is consistently reproducible and is compatible with an interaction of a small peptide like ANG II (1 kDa) with a large protein like megalin (600 kDa). The converse experiment binding the large megalin molecule to immobilized ANG II is precluded by the expense of purifying sufficient megalin while maintaining binding activity.4 t3 I% {* Z( I/ q

Interference with ANG II uptake by both candesartan and IP shows significant but very partial inhibition. The CP also significantly inhibited ANG II uptake, although the IP is greater. This effect could be explained by the fact that CP was designed as the reverse sequence of IP to retain many of the charge distribution properties of the active peptide intact. None of the peptide data on AT1R-mediated ANG II uptake distracts from the central finding that this receptor is only responsible for a small fraction of the uptake, and several lines of evidence presented here implicate megalin as the predominant ANG II uptake pathway." [: T0 `. M9 U3 F

The coexpression of megalin and the AT1R, in epithelial tissues like the PT, might be important in the regulation of metabolism and function of ANG II. The PT is normally exposed to high levels of ANG II because the concentration of ANG II in the tubular fluid bathing this segment is 6–10 nM (36). These concentrations are higher than the kilodaltons of the AT1R (43) and are inappropriately maintained in chronically infused ANG II animals (48). This suggests that a protective and compensatory mechanism operates to maintain intracellular ANG II in the PT within a certain range. In Fig. 5, we present a hypothetical model that would explain the role of megalin in such a mechanism. We hypothesize that in the PT and perhaps other tissues that coexpress both receptors, under physiological conditions at least two mechanisms interact to maintain intracellular ANG II levels in a constant range. The first, mediated by AT1R, leads to ANG II accumulation and helps to regulate several functions and intracellular pathways in the PT as has been suggested in the past (4, 5, 40, 41). This pathway might also shuttle ANG II to intracellular sites (30). The second mechanism is mediated by megalin and serves the PT as a scavenger pathway. That is, it targets ANG II to degradation, protecting the cell against ANG II accumulation. The balance between these two pathways will ultimately determine intracellular ANG II levels.! n9 p1 ]  j2 X/ L) D% q
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Zou et al. (54) demonstrated that after chronic ANG II administration in rats, there is an accumulation of ANG II in the whole kidney that is prevented by the concomitatnt treatment with ARBs, findings supported by experiments performed in other animal models (46). Zhuo et al. (53) also demonstrated that ANG II content was higher in endosome and intermicrovillar cleft vesicles extracted from kidney cortexes of ANG II chronically infused rats than of controls. A low-salt diet also raises circulating and kidney levels of ANG II (22, 24). The latter can be prevented as well by AT1R blockade. However, Imig et al. (22) found no differences in either the endosomal or the intramicrovillar cleft content between animals fed with different levels of NaCl content in the diet. Thus, as a difference with ANG II-infused animals, an increase in ANG II content in intracellular compartments extracted mainly from the PT was expected but not observed in animals on a low-salt diet, even though the kidney concentration and AT1R expression were higher (22). We think that the difference between these two groups (ANG II infused vs. low-salt diet) can be at least partly explained by megalin expression because recent evidence demonstrates that ANG II-infused animals exhibit downregulation of megalin expression and function in the PT (45).# V) |1 l8 o- l4 W/ [$ W/ c% C

Under nonphysiological conditions, however, as might occur with chronic ANG II infusion, several factors may contribute to disrupt the equilibrium between megalin and the AT1R pathways, causing upregulation of the AT1R and/or downregulation of megalin uptake (Fig. 5B). During ANG II infusion, there is upregulation of the AT1R (22, 23) and some other components of the tubular RAS, like angiotensinogen (23, 28), representing a positive feedback in the tubular RAS system and ultimately more production of ANG II. In addition, as previously mentioned, chronic ANG II infusion causes downregulation of megalin expression and function in the PT (45). There is also evidence of cross talk between these two pathways, because the detrimental effect of ANG II infusion on megalin expression can be reversed in vivo by the use of angiotensin-converting enzyme inhibitors or ARBs (45). This cross talk can be further explained by the effects of ANG II on transforming growth factor- (TGF-) due to the fact that ANG II induces TGF- (50) and TGF- type II receptor expression (51), with the activation of the TGF-1 pathway, leads to reduced megalin/cubilin-mediated endocytosis, via TGF-/Smad-dependent expression (16). Additionally, ANG II binding to megalin might trigger megalin proteolysis and activation of separate pathways, a role that has been suggested recently for megalin (55). Finally, megalin could in theory mediate the transport of ANG II to intracellular sites or its transocytosis [as is the case of the transferrin receptor (11)].# \  p: T' q1 Q5 h9 d. x

So far, the major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of the known ligands (15). The role of megalin as a receptor for ANG II suggests that megalin mediates physiological and pathophysiological mechanisms linked to endogenous ligands in vivo and raises important questions regarding the interactions between megalin and the renal tubular RAS in the physiology of the kidney, blood pressure, and salt and water regulation.
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In summary, evidence obtained from cell lines and animal preparations supports the role of megalin and perhaps cubilin as mediators of the binding and uptake of ANG II. This putative alternate pathway is quantitatively more important than AT1R for short-term ANG II uptake at least in placental epithelial cells.  u7 N8 S& ~* d9 k5 {  K0 T" F$ `
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GRANTS
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This work was supported by National Institute of Environmental Health Sciences Advanced Research Cooperation in Environmental Health Grant ES-09996 (to R. B. S. Klassen and T. G. Hammond) and National Heart, Lung, and Blood Institute Grant HL-26371 (to L. G. Navar). We thank Veterans Affairs, New Orleans, for providing space, equipment, and salaries (T. G. Hammond) in support of these studies. Flow cytometry equipment was purchased under the auspices of a grant from the Louisiana Educational Quality Support Fund from the Board of Regents. Additional support was provided by National Institutes of Health Grant P20-RR-017659 from the Institutional Development Award Program of the National Center for Research Resources and by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund (2001-06)-07./ G' @4 {" Y* S& @. x: ]& y* A
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FOOTNOTES
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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.
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