Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, Hiroshima 734-8551, Japan
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ABSTRACT |
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The role of megalin in tissue distribution of aminoglycosides was examined in normal rats and maleate-treated rats that shed megalin from the renal brush-border membrane. In normal rats, amikacin administered intravenously accumulated most abundantly in the renal cortex, followed by the renal medulla. No amikacin was detected in other tissues. Tissue distributions of amikacin were well correlated with megalin levels in each tissue. Bolus administration of gentamicin increased urinary excretion of megalin ligands (vitamin D binding protein and calcium), suggesting the competition between gentamicin and these megalin ligands in renal tubules. Ligand blotting showed that binding of 45Ca2+ to megalin was inhibited by aminoglycosides. Both megalin levels and amikacin accumulation in renal cortex were decreased by maleate injection. Then, amikacin accumulation recovered proportionate to megalin levels. These findings suggest that megalin is involved in the renal cortical accumulation of aminoglycosides in vivo. In addition, the interaction between aminoglycosides and calcium in the kidney may be due to the competition among these compounds to bind to megalin.
receptor-meditated endocytosis; tissue distribution; polybasic drugs; calcium; vitamin D binding protein
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INTRODUCTION |
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NEPHROTOXICITY IS A MAJOR clinical complication of aminoglycoside antibiotics, which are widely used for the treatment of Gram-negative infectious disease (27, 35). Aminoglycosides are not metabolized in the body, and most of the injected dose is excreted into the urine, whereas a fraction accumulates in the renal proximal tubular cells, where the concentration of aminoglycosides is several times higher than in plasma. The concentrated accumulation of aminoglycosides in the proximal tubular cells is associated with the nephrotoxic effects (14). The transport of aminoglycosides in the kidney has been studied by various techniques in vivo (12, 16, 17, 30) and in vitro (5, 13, 18, 23, 38). These results indicate that aminoglycosides interact with the brush-border membrane in the renal proximal tubular cells and are subsequently taken up by an adsorptive endocytosis. In addition, acidic phospholipids, like phosphatidylinositol, are thought to be the binding sites of aminoglycosides in the brush-border membrane (10, 36, 37). On the other hand, because acidic phospholipids are commonly distributed in the plasma membranes in various tissues, other factors mediating endocytosis of aminoglycosides, specifically in the renal proximal tubular cells, have been assumed to be involved. However, the membrane receptor involved in the binding and endocytosis of aminoglycosides in the kidney has not been identified until recently.
Megalin, initially identified as a Heymann nephritis autoantigen
(19, 34), is a large glycoprotein (~600 kDa),
functioning as an endocytic receptor. Now also considered a low-density
lipoprotein receptor-related protein-2 (LRP-2), megalin is a member of
the low-density lipoprotein receptor gene family and is expressed in
the renal proximal tubule epithelium, labyrinth epithelium of the ear,
retinal epithelium, type II pneumocytes, and yolk sac
(40). The cytoplasmic tail of megalin contains the NPXY sequence, which is necessary for coated pit-mediated internalization (3, 34). The ligands binding to megalin represent a
variety of classes including Ca2+ (4),
lipoprotein lipase (21), plasminogen activator-plasminogen activator inhibitor type-1 complexes (26), and
receptor-associated protein (RAP) (20). Recently, Nykjaer
et al. (28) found that complexes of 25-(OH) vitamin
D3 and vitamin D binding protein (DBP) filtered through the
glomerulus are taken up by megalin into the proximal tubular cells,
where 25-(OH) vitamin D3 is further metabolized to the
physiologically active 1,25-(OH)2 vitamin D3. In addition, megalin would also be involved in the
binding and endocytosis of polybasic drugs such as aminoglycosides,
aprotinin, and polymixin B in the renal brush-border membrane
(25). The rank order of the apparent affinity of various
aminoglycosides for purified megalin agrees with that of their
nephrotoxic potentials. However, the role of megalin in the renal
handling of these ligands in vivo needs further clarification.
In the present study, we analyzed the expression of megalin and the distribution of aminoglycosides in tissues of normal and maleate-treated rats. Our results suggest that megalin is involved in the accumulation of aminoglycosides in the renal cortex.
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MATERIALS AND METHODS |
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Materials. The following reagents were used: amikacin sulfate salt (Sigma, St. Louis, MO), maleic acid disodium salt, gentamicin sulfate, 2,4,6-trinitrobenzenesulfonic acid sodium salt, polymixin B sulfate (Nacalai Tesque, Kyoto, Japan), and inulin (Kanto Chemical, Tokyo, Japan). 45Ca2+ (0.72 GBq/mg calcium) was obtained from Amersham International (Buckinghamshire, UK). All chemicals used were of the highest purity available.
Preparation of antiserum against megalin. Rabbit anti-megalin antibody was raised against the synthetic peptide corresponding to the 17-amino acid peptide for the cytoplasmic domain of rat megalin (MEVGKQPVIFENPMYAA), with an additional cystein at the NH2 terminal (1, 34). The synthetic peptide was produced with the peptide synthesizer (PSSM-8, Shimadzu). The peptide was linked to a carrier protein, keyhole limpet hemocyanin (Sigma), with a coupling agent, m-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma). The cross-linked peptide was mixed with an equal volume of Freund's complete adjuvant (Wako Pure Chemicals, Osaka, Japan), and the emulsion was injected intradermally into New Zealand White female rabbits (2.0-2.5 kg) on day 0. Subsequent immunization was performed with incomplete Freund's adjuvant (Wako Pure Chemicals) at four intervals between days 28 and 64. Serum samples were tested for reactivity with the antigen peptide by dot-blot analysis, using a horseradish peroxidase-conjugated goat antibody to rabbit IgG (ICN Pharmaceuticals, Aurora, OH) as a secondary antibody. At day 36 after the start of immunization, antibodies reactive with the antigen peptide were detected by dot-blot analysis.
Western blot analysis and ligand blotting. Excised tissues were homogenized in 1% Triton X-100 in 20 mM Tris, 150 mM NaCl and centrifuged at 14,000 g for 15 min in an Avanti 30 compact centrifuge with rotor F0630 (Beckman Instruments). The supernatant was solubilized in a loading buffer; 2% SDS, 62.5 mM Tris · HCl, 7% glycerol with or without 5% 2-mercaptoethanol (reducing or nonreducing condition, respectively). Collected urine was dialyzed by use of cellulose membrane and was concentrated by freeze-drying. These samples were subjected to SDS-PAGE with polyacrylamide gels of 6% for megalin detection or 10% for DBP detection. The proteins were transferred for 60 min to polyvinylidene difluoride membrane (Immun-Blot, Bio-Rad) at 4°C. In Western blot analysis, the membrane was blocked in 5% nonfat dry milk in Tris-buffered saline (TBS; 150 mM NaCl, 20 mM Tris · HCl, pH 7.5) with 0.05% Tween 20 (TBS-T) overnight at 4°C. The membranes were washed three times for 10 min each in TBS-T and incubated with the rabbit antiserum to the cytoplasmic tail of rat megalin (1: 5,000 dilution) or anti-human DBP rabbit antiserum (1:100 dilution) purchased from DAKO Japan (Kyoto, Japan). The membranes were washed three times in TBS-T and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000 dilution, ICN Pharmaceuticals), again washed three times in TBS-T, and visualized with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK). Ligand blotting with 45Ca2+ was carried out as described by Maruyama et al. (24). Briefly, the membranes were washed with a buffer containing 60 mM KCl, 5 mM MgCl2 and 10 mM imidazole-HCl (pH 6.8), and the buffer was changed three times in 1 h. Subsequently, the membranes were incubated for 10 min in the same buffer containing 45Ca2+ (37 MBq/l) in the absence or presence of various cationic compounds. After being rinsed with 50% ethanol for 5 min, the membranes were dried at room temperature for 3 h. Autoradiograms of the 45Ca2+-labeled proteins were obtained by exposure of the dried membranes to Amersham Hyperfilm for 24-72 h.
Animal experiments. Experiments with animals were performed in accordance with the Guide for Animal Experimentation, Hiroshima University, and the Committee of Research Facilities for Laboratory Animal Sciences, School of Medicine, Hiroshima University. To induce the shedding of megalin from the renal brush-border membrane, male Wistar rats (260-320 g) received sodium maleate (400 mg/kg, pH 7.4) by intraperitoneal injection. Rats injected with the same volume of saline were used as control. Subsequently, the rats were anesthetized with pentobarbital sodium (30 mg/kg), and the femoral artery and vein were cannulated with polyethylene tubing for blood sampling and drug administration, respectively. The urinary bladder was cannulated to collect the urine sample. At a stated time after the maleate injection, amikacin (3.5 mg/kg) and inulin (20 mg/kg) were administrated as a bolus via the femoral vein. At 120 min after the administration of amikacin, each tissue was excised. The tissues were weighed and then homogenized with 4 vol of ice-cold 3% perchloric acid. After centrifugation at 3,000 rpm for 15 min, amikacin concentration in the supernatant was assayed as described below.
Analytic methods. Quantitative determination of amikacin was performed by using high-performance liquid chromatography (PU-980, Jasco, Tokyo, Japan) equipped with an ultraviolet spectrophotometric detector (UV-970, Jasco; wavelength 229 nm) after derivatization of the drug by reaction with 2,4,6-trinitrobenzenesulfonic acid (11). The conditions were as follows: column 4.6 × 100 mm, TSK gel ODS-80TM (Tosoh, Tokyo, Japan); mobile phase 0.02 M phosphate buffer (pH 7.5)-acetonitrile-methanol (vol/vol) = 10:11:5; flow rate 1.0 ml/min; and room temperature. Inulin concentrations in the urine were analyzed by spectrophotometric assay as stated previously (22). Urinary calcium was measured with commercial kits available from Wako Pure Chemicals.
Statistical analysis was performed by Student's t-test. A difference of P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Expression of megalin and accumulation of amikacin in various
tissues in normal rats.
First, we characterized the rabbit antiserum raised against the
synthetic peptide for the cytoplasmic domain of rat megalin. As shown
in Fig. 1c, the antiserum
detected a high-molecular-weight protein (>400 kDa) in rat renal
cortex protein subjected to gel electrophoresis. A band of the same
size was observed by blotting with 45Ca2+ (Fig.
1e), which disappeared when reduced with 2-mercaptoethanol (data not shown). The band was not detected when the preimmune serum
was used (Fig. 1b). In addition, the band detected by the antiserum disappeared in the presence of the antigen peptide (Fig. 1d). Thus the anti-cytoplasmic peptide antiserum we obtained
was shown to specifically recognize megalin. With this anti-megalin antiserum, the tissue distribution of megalin was examined in normal
rats by Western blot analysis and immunohistochemical analysis. As
shown in Fig. 2, the expression of
megalin was highest in the renal cortex. The megalin band was also
detected in the renal medulla but was not observed in the renal
papilla, brain, lung, or liver. Furthermore, the immunohistochemical
study showed that megalin was located in the apical membrane of the
proximal tubules in rat renal cortex, whereas no staining was observed
in the liver (data not shown). Next, the tissue distribution of
amikacin was examined 120 min after an intravenous bolus injection of
the drug (Fig. 3). Amikacin was more
abundant in the renal cortex than other tissues examined. Amikacin was
found to a lesser extent in the renal medulla but was not detected in
the renal papilla, brain, or liver. Thus the tissue distribution of
amikacin was well correlated with the expression level of megalin.
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Interaction between aminoglycosides and megalin ligands.
The effect of intravenous bolus administration of gentamicin on urinary
excretion of such endogenous megalin ligands as DBP and calcium was
examined. The administration of gentamicin (3.5 mg/kg) increased the
urinary excretion of DBP (Fig. 4).
Urinary calcium was also significantly increased by the gentamicin
administration (control, 46.1 ± 17.8 µg/120 min,
n = 5; gentamicin-administration, 119.8 ± 23.8 µg/120 min, n = 3, means ± SE,
P < 0.05). To analyze the involvement of megalin in
calcium-aminoglycoside interaction, we examined the effect of
aminoglycosides on the binding of 45Ca2+ to
megalin in the renal cortex (Fig. 5).
Unlabeled calcium (2 mM) almost completely inhibited the binding of
45Ca2+ to megalin.
45Ca2+ binding to megalin was inhibited by the
presence of aminoglycosides (gentamicin, amikacin). In addition, the
binding of 45Ca2+ was inhibited by polymixin B,
a basic peptide antibiotic, which was reported to be a ligand of
megalin by Moestrup et al. (25).
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Effect of maleate administration on megalin level and
amikacin accumulation in renal cortex.
Bergeron et al. (2) have reported that maleate in
rats induces shedding of megalin from the brush-border membrane in the renal proximal tubule cells and increases urinary excretion of megalin.
Therefore, maleate-treated rats may be useful as model animals with
which to examine the in vivo function of megalin in the renal proximal
tubules. To confirm the decrease in megalin in the renal proximal
tubules by the administration of maleate in the present study, megalin
levels in the renal cortex and in the urine from maleate-treated rats
were examined by Western blot analysis and by ligand blotting with
45Ca2+. Similar results were obtained by using
both methods: a decrease in megalin level in the renal cortex and the
appearance of megalin in the urine of maleate-treated rats (Fig.
6, A and B). Then, renal accumulation of intravenously administered amikacin was examined
in normal rats and rats treated with maleate. The results showed that
the amount of amikacin accumulated in the renal cortex of
maleate-treated rats was significantly lower than that of normal rats (Fig. 7A). The decrease
in the amikacin accumulation in the renal cortex would not be due to
the change in the amount of amikacin filtered through the glomerulus,
because the urinary excretion of amikacin for 120 min (Fig.
7B), as well as that of inulin (data not shown), was not
changed by the maleate treatment. Furthermore, the relationship between
amikacin and megalin levels in the renal cortex was examined at various
intervals after maleate injection. The amikacin accumulation in the
renal cortex recovered with time in accordance with the recovery of
megalin levels in the renal cortex (Fig.
8, A and
B).
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DISCUSSION |
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Aminoglycosides are low-protein binding drugs (<10%), freely filtered through the glomerulus in the kidney. Subsequently, aminoglycosides are selectively accumulated and remain, with a long half-life, in the renal cortex (35). Many studies have shown that the uptake of aminoglycosides in the renal proximal tubules is due to an endocytic system, such as a receptor-mediated endocytosis. However, little information is available on the endocytic receptor. Recently, Moestrup et al. (25) reported that polybasic drugs including aminoglycosides bind to purified megalin, which is an endocytic receptor abundantly expressed in the renal proximal tubules. In addition, they found that RAP inhibits the uptake of [3H]gentamicin microperfused into single proximal tubules in rat. These observations suggest the possibility that megalin is involved in the accumulation of aminoglycosides in the renal cortex. However, it has not been demonstrated that megalin plays an important role in the tissue distribution of aminoglycosides in vivo. In the present study, we examined the in vivo role of megalin in the tissue distribution of aminoglycosides using normal and maleate-treated rats.
The present results showed that amikacin administered intravenously was accumulated most abundantly in the renal cortex (Fig. 3), where the expression of megalin was highest among various tissues examined (Fig. 2). On the other hand, amikacin was not detected in the renal papilla, brain, lung, and liver (Fig. 3). Zheng et al. (40) detected megalin in brain ependyma and lung pneumocytes type II in an immunohistochemical study, but we did not detect megalin by Western blot analysis using whole tissue homogenates of the brain and lung. Megalin expressed in these specialized regions of the brain and lung might be involved in the endocytosis of megalin ligands in cerebrospinal fluid and respiratory airways, where intravenously administered amikacin is unlikely to be distributed. Therefore, we did not detect amikacin in the brain and lung because of the insensitivity of our analytic method, low expression of megalin in these tissues, or the limited distribution of intravenously administered amikacin where megalin is expressed.
Interestingly, aminoglycosides such as gentamicin, netilmicin, and amikacin are reported to inhibit in vitro binding of the 125I-urokinase-plasminogen activator inhibitor-1 complex not only to megalin but also to LRP, a member of the low-density lipoprotein receptor gene family (25), suggesting the interaction between aminoglycosides and LRP. The expression pattern for LRP differs strikingly from that of megalin. In contrast to megalin, LRP is abundantly expressed in the liver but not in the renal proximal tubules (40). However, as shown in this study, amikacin was not accumulated in the liver in vivo (Fig. 3), suggesting that LRP does not play an important role in the tissue uptake of aminoglycosides. The reason for this apparent discrepancy between in vitro and in vivo observations is presently unknown. In addition to megalin, other factors may be involved in the selective accumulation of aminoglycosides in the kidney.
Maleate is often used to induce the experimental Fanconi syndrome (2, 32, 33). In the present study, megalin levels in normal and maleate-treated rats were estimated by two methods: ligand blotting with 45Ca2+, which binds to the extracellular domain of megalin, and Western blotting with a rabbit antiserum raised against the synthetic peptide for the cytoplasmic domain of megalin. These two methods revealed a decrease in megalin level in the renal cortex of maleate-treated rats. In addition, megalin was detected in the urine of maleate-treated, but not of normal, rats (Fig. 6). These results indicate that megalin excreted in the urine of maleate-treated rats would retain the cytoplasmic domain (without cleavage in the ectodomain). This is consistent with the previous report that a polyclonal antibody against the cytoplasmic tail of megalin showed a mosaic pattern of megalin staining in the renal tubules in maleate-treated rats (2). However, it is unclear how megalin retaining the cytoplasmic domain sheds from the brush-border membrane by maleate administration.
The administration of maleate reduced the renal accumulation of amikacin, corresponding to the decrease in megalin level in the renal cortex (Fig. 7). Furthermore, we found that amikacin accumulation in the renal cortex of maleate-treated rats recovered over time in accordance with the recovery of megalin level in the renal cortex, suggesting that the renal cortical accumulation of amikacin is dependent on the megalin level in the renal cortex (Fig. 8). There are several previous reports showing that maleate administration induces the decrease in the reabsorption of aprotinin and lysozyme in the renal proximal tubules (9, 39). Recently, both aprotinin and lysozyme are shown to be ligands of megalin (25, 29). These observations as well as the present results suggest that megalin is involved in the in vivo reabsorption of these cationic compounds. However, the involvement of a nonspecific effect of maleate on endocytosis in the proximal tubules cannot be ruled out.
If megalin plays an important role in the renal uptake of
aminoglycosides as described above, the administration of
aminoglycosides may induce the increase in urinary excretion of
endogenous megalin ligands due to the competitive inhibition of the
binding to megalin. To test this possibility, the effect of gentamicin
injection was examined. As was expected, the intravenous injection of
gentamicin increased the urinary excretion of DBP (Fig. 4) and calcium,
both of which are ligands of megalin (4, 28). Consistent
with the increase in urinary calcium by the administration of
gentamicin in vivo, the binding of 45Ca2+ to
megalin blotted on the filter membrane was inhibited by gentamicin and
amikacin (Fig. 5). In addition, polymixin B, which has a relatively high affinity for megalin (25), also decreased the calcium
binding to megalin. Some reports have focused on the interaction
between aminoglycosides and calcium in the kidney. Humes et al.
(15) reported that calcium inhibits the binding of
aminoglycosides to the renal plasma membranes and that oral calcium
loading protects against aminoglycoside nephrotoxicity. Quarum et al.
(31) also found that dietary calcium loading attenuates
the severity of gentamicin nephrotoxicity in rats. In addition, it has
been shown that the hypercalciurea induced by low doses of gentamicin
occurs without accompanying nephrotoxicity (7, 8).
Moreover, injection of a single, standard clinical dose of gentamicin
was shown to produce an immediate and transient increase in urinary
excretion of calcium in healthy human subjects (6). Taken
together, these findings indicate that megalin is involved in various
interactions observed between aminoglycosides and calcium, such as
calciuresis induced by aminoglycosides, in the kidney. In addition,
inhibition of the renal uptake of DBP by gentamicin may also affect
calcium homeostasis, because DBP is essential for the transport of
25-(OH) vitamin D3 into the proximal tubular cells, where
25-(OH) vitamin D3 is changed to 1,25-(OH)2
vitamin D3, a steroid hormone important in the regulation
of systemic calcium and bone metabolism.
In conclusion, the present study shows that megalin would be responsible for the aminoglycoside accumulation in the renal cortex under in vivo condition. In addition, it is suggested that the interaction between aminoglycosides and calcium in the kidney is partly due to the competition of these compounds for binding to megalin.
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ACKNOWLEDGEMENTS |
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We thank Dr. Hiroki Kuniyasu (First Dept. of Pathology, Faculty of Medicine, Hiroshima University) for his assistance with the immunolocalization study. We also thank the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University, for the use of their facilities.
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FOOTNOTES |
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan and in part by the Sankyo Foundation of Life Science.
Address for reprint requests and other correspondence: M. Takano, Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima Univ., 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan (E-mail address: takanom{at}pharm.hiroshima-u.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.
Received 10 July 2000; accepted in final form 20 April 2001.
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