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Targeted prevention of renal accumulation and toxicity of gentamicin by aminoglycoside binding receptor antagonists
Journal of Controlled Release 95 (2004) 423 – 433 www.elsevier.com/locate/jconrel
Targeted prevention of renal accumulation and toxicity of gentamicin by aminoglycoside binding receptor antagonists Ayahisa Watanabe, Junya Nagai, Yoshinori Adachi, Takayuki Katsube, Yasumi Kitahara, Teruo Murakami, Mikihisa Takano * Department of Pharmaceutics and Therapeutics, Division of Clinical Pharmaceutical Science, Programs for Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Received 2 September 2003; accepted 9 December 2003
Abstract Receptor-mediated endocytosis plays an important role in accumulation of aminoglycosides in renal proximal tubule. To prevent aminoglycoside-induced nephrotoxicity following concentrated accumulation of gentamicin in the kidney, effect of cationic proteins and their peptide fragments, which could inhibit gentamicin binding to its binding receptor(s), was investigated. Among several substrates for megalin, an endocytic receptor responsible for renal accumulation of aminoglycosides, cytochrome c potently inhibited gentamicin accumulation in renal cortex. Concentration-dependent inhibition by cytochrome c on gentamicin uptake was also observed in OK kidney epithelial cells expressing megalin. In addition, gentamicin-induced increase in urinary excretion of N-acetyl-h-D-glucosaminidase (NAG), a marker of renal tubular damage, was significantly reduced by cytochrome c. We next attempted to find a peptide fragment with lower molecular size showing inhibitory effect on gentamicin uptake. Cyto79-88 inhibited gentamicin uptake in OK cells, but had little effect on renal accumulation of gentamicin in mice in vivo. On one hand, a peptide fragment of neural Wiskott – Aldrich syndrome protein (N-WASP), which interacts with acidic phospholipids like aminoglycosides, inhibited gentamicin accumulation not only in OK cells but also in mouse kidney. These results show that substrates and/or their peptide fragments for aminoglycoside binding receptor such as megalin might be useful for preventing aminoglycoside-induced nephrotoxicity. D 2004 Elsevier B.V. All rights reserved. Keywords: Aminoglycosides; Nephrotoxicity; Receptor-mediated endocytosis; Megalin; Acidic phospholipids
1. Introduction Aminoglycoside antibiotics are widely used for the treatment of gram-negative infections. However, nephrotoxicity remains a common clinical problem and its toxicity is one of dose-limiting factors in the * Corresponding author. Tel.: +81-82-257-5315; fax: +81-82257-5319. E-mail address: [email protected] (M. Takano). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.12.005
use of aminoglycosides. Reportedly, the average frequencies of nephrotoxicity for gentamicin and tobramycin were 14.0% and 12.9%, respectively [1], whereas it has been also reported that the nephrotoxicity rates range from 1.7% to 58% depending on the definition [2]. In any case, aminoglycoside-induced nephrotoxicity appears to be directly related to the concentrated accumulation of aminoglycosides in the renal proximal tubular cells. Therefore, over the past few decades, a considerable number of studies have
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been made on the pathway responsible for aminoglycoside uptake in the renal proximal tubular cells [3– 5]. Most of aminoglycoside injected into the body is excreted into the urine without being metabolized. However, the rest of the injected dose accumulates selectively and abundantly in the renal cortex. Aminoglycoside taken up by the renal proximal tubular cells stays there for a long time, leading to renal damage such as structural change and functional impairment of plasma membrane, mitochondria and lysosome [4]. Consequently, there has been a great interest in the mechanism with which aminoglycosides are absorbed into the renal proximal tubular cells across the brushborder membrane. So far, many results have indicated that aminoglycosides are taken up by receptor-mediated endocytosis following the binding of aminoglycosides to the brush-border membrane. Sastrasinh et al. [6] suggested the acidic phospholipids as a binding site for aminoglycosides in rat renal brush-border membrane. In the paper, they showed that [3H]gentamicin binds in a saturable manner to several phospholipids such as phosphatidylinositol4,5-bisphosphate (PIP2), phosphatidic acid, phosphatidyl inositol, phosphatidylserine and phosphatidylinositol-4-monophosphate (PIP) [6]. More recently, it has been suggested that megalin, an endocytic receptor expressed at the apical membrane of renal proximal tubules [7 –9], plays an important role in binding and endocytosis of aminoglycosides in renal proximal tubular cells [10 –12]. Megalin belonging to the lowdensity lipoprotein (LDL) receptor family has been shown to bind Ca2 +, vitamin binding proteins such as vitamin D-binding protein and retinal-binding protein, lipoproteins such as apolipoprotein E and apolipoprotein H, enzymes such as lipoprotein lipase and lysozyme, cytochrome c, hemoglobin, drugs such as aminoglycosides, polymixin B, aprotinin, and so on [8,13,14]. Moestrup et al. [10] found that receptorassociated protein (RAP), one of common ligands for LDL receptors, inhibited [3H]gentamicin uptake in perfused rat proximal tubules. In addition, we observed that the intravenous administration of gentamicin to rat increased the urinary excretion of endogenous vitamin D-binding protein [11] and fluorescein isothiocyanate-labeled lysozyme [15]. These results indicate an interaction between gentamicin and megalin substrates at the apical membrane of the renal
proximal tubular cells. Therefore, megalin may be a useful target for attenuating aminoglycoside-induced nephrotoxicity. Actin-regulating proteins such as gelsolin, CapG and neural Wiskott – Aldrich syndrome protein (NWASP) have been reported to have a phosphoinositide-binding region which contains several basic amino acids like megalin substrates. Recently, some phosphoinositide-binding sequences in their actinregulating proteins have been identified [16 – 18]. Since phophoinositides such as PIP2 are also suggested to serve as a receptor responsible for aminoglycoside accumulation in the kidney as described above, a peptide fragment derived from actin-regulating protein, as well as that from a substrate of megalin, may be useful for preventing aminoglycoside-induced nephrotoxicity. In the present study, we demonstrate that megalin substrates such as cytochrome c prevent gentamicininduced nephrotoxicity as well as the accumulation of gentamicin in the renal cortex in rats, probably by decreasing the binding of gentamicin to megalin. Furthermore, cationic peptide fragments, which may interact with anionic phospholipids as well as megalin, significantly inhibited the accumulation of gentamicin in OK cells and in mouse kidney.
2. Materials and methods 2.1. Materials [3H]Gentamicin sulfate (7.4 GBq/g) was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). Lysozyme chloride from egg white, aprotinin from bovine lung and gentamicin sulfate were obtained from Nacalai Tesque (Kyoto, Japan). Cytochrome c from bovine heart (MW = 12,327) was purchased from Sigma Chemical (St. Louis, MO, USA). N-Acetyl-h-D-glucosaminidase (NAG) assay kit was from Shionogi and Co. (Osaka, Japan). All other chemicals used for the experiments were of the highest purity available. 2.2. Preparation of peptide fragments Synthetic peptide fragments were produced with the peptide synthesizer (PSSM-8, Shimazu). The peptide
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fragments were synthesized chemically and their amino acid sequences were as follows: Cyto22-41 (KGGKHKTGPNLHGLFGRKTG), Cyto70-89 (NPKKYIPGTKMIFAGIKKKG), Cyto79-88 (KMIFAGIKKK), N-WASP181-200 (NISHTKEKKKGKAKKKKRLTK), Gelsolin150-169 (KHVVPNEVVVQRLFQVKGRR), and CapG137-146 (KKLYQVKGKK). The peptide sequence of the fragments prepared by the synthesizer was confirmed by matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (PerSeptive Biosystems, Tokyo, Japan). 2.3. In vivo uptake study 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, Graduate School of Biomedical Sciences, Hiroshima University. In rat study, male Wistar rats (230 – 290 g) were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg) and the femoral artery and vein were cannulated with polyethylene tubing for blood sampling and drug administration. The animals were placed on a heating pad to maintain body temperature at 37 jC. [3H]Gentamicin (25 Ag per rat) without or with a candidate antagonist was injected into a femoral vein. Blood samples (50 Al) were withdrawn through the femoral artery at the specified time (1, 5, 10, 20, 30, 45, 60, 90 and 120 min). In mouse study, male ddy mice (22 –28 g) were administered [3H]gentamicin (85 Ag/kg) alone or in combination with a candidate antagonist via the tail vein. Blood samples of mice were obtained by cardiac puncture. At 120 min after the administration into rats or mice, kidneys were excised. In the case of rat study, the renal cortex was dissected from the excised kidney by the use of a Stadie-Riggs microtome. In mouse study, the whole kidney was used to examine the accumulation of [3H]gentamicin. The tissues were weighed and homogenized with 0.25 M sucrose. An aliquot of the homogenates (100 Al) was solubilized with 400 Al of NCSII (Amersham Pharmacia Biotech, Buckinghamshire, UK). Subsequently, 5 ml of ACSII (Amersham Pharmacia Biotech) was added and the radioactivity was measured by liquid scintillation counting.
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2.4. Evaluation of nephrotoxicity Male Wistar rats (210 – 270 g) were maintained without water for 48 h to produce dehydrated rats which are much more susceptible to aminoglycosideinduced renal failure as compared with normal rats [19,20]. The dehydrated rats were injected intravenously with saline (control), 30 mg/kg gentamicin alone (GM), or 30 mg/kg gentamicin with 100 mg/ kg cytochrome c (GM + CYT c). Then, the animals were kept further without water for 17 h. Thereafter, the rats were housed in metabolic cages to collect urine for 24 h with free access to water. NAG in the urine was measured spectrophotometrically using a commercially available kit (Shionogi and Co.). 2.5. Cell culture OK cells were cultured in medium 199 (Gibco BRL, Life Technologies, NY, USA) containing 10% fetal bovine serum (Biological Industries, Israel) without antibiotics, in an atmosphere of 5% CO2 – 95% air at 37 jC, and subcultured every 6 or 7 days using 0.02% EDTA and 0.05% trypsin as described previously [21]. OK cells were used between passages 85 and 98. 2.6. In vitro uptake study Uptake of [3H]gentamicin was measured in OK cells attached to the 12-well plates. Briefly, fresh medium was replaced every 2 or 3 days, and the cells were used on the sixth or seventh days after seeding. Experiments were performed in Dulbecco’s phosphate-buffered saline (PBS buffer containing in mM, 137 NaCl, 3 KCl, 8 Na2HPO4, 1.5 KH2PO4, 0.1 CaCl2 and 0.5 MgCl2) supplemented with 5 mM Dglucose (PBS (G) buffer). After removal of the culture medium, each dish was washed and preincubated with PBS (G) buffer. Then, PBS (G) buffer containing [3H]gentamicin in the absence or presence of a candidate antagonist was added to each dish and the cells were incubated at 37 jC for a specified period. At the end of the incubation, the uptake buffer was aspirated and the dishes were rinsed rapidly three times with 1 ml of ice-cold PBS buffer. The cells were scraped with rubber policeman and then were washed by centrifugation at 4 jC for 1 min at 10,000 rpm twice. After the
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supernatant was aspirated, the pellet was solubilized in 0.1 M NaOH, and the amount of substrate taken up by the cells was measured by counting the radioactivity. Protein was determined by the method of Bradford [22] with bovine serum albumin as the standard. 2.7. Western blot analysis and ligand blotting For immunoblot analysis of megalin in OK cells, the crude membrane fraction from OK cells was prepared the sixth day after seeding as described previously [23]. Briefly, after removal of the culture medium, each dish was washed with ice-cold PBS buffer and the cells were collected with rubber policeman. The cell suspension was homogenized for 2 min with an IKA T25 Basic disperser (IKAR LABORTECHNIK, Germany) in an ice-cold buffer (150 mM NaCl, 1 mM, EDTA, 1 mM PMSF with 20 mM Tris, pH 7.4), and was subsequently homogenized with a glass/Teflon Potter homogenizer with 10 strokes at 1000 rpm. The homogenate was centrifuged at 3000 g for 10 min at 4 jC in an Avanti 30 Compact Centrifuge with rotor F0630. The supernatant was centrifuged at 40,000 g for 30 min at 4 jC. The pellet was resuspended in the ice-cold buffer containing 1% Triton X-100, and centrifuged at 14,000 g for 15 min at 4 jC. The supernatant, which contains crude membrane fractions of OK cells, was heated for 5 min at 95 jC in a loading buffer. The crude membrane fractions of rat renal cortex for control sample of megalin detection were prepared as described in the previous paper [21]. These samples were subjected to SDS-polyacrylamide gel electrophoresis with 6% polyacrylamide gels, and the proteins were transferred for 60 min to PVDF membrane at 4 jC. The membrane was blocked in 5% non-fat dry milk in TBS-T overnight at 4 jC. The membranes were washed three times for 10 min in TBS-T, and were incubated with the anti-rat megalin rabbit antiserum (1:5000 dilution) [11]. The membranes were washed three times in TBS-T, and were incubated with the horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2000 dilution), washed three times in TBS-T, and visualized with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Ligand blotting with 45Ca2 + was carried out as described previously [11,13].
2.8. Data analysis Statistical analysis was performed by Student’s t-test, or by the one way analysis of variance (ANOVA) with the Scheffe´ test for post hoc analysis. A difference of P < 0.05 was considered statistically significant. The isoelectric points (pI) of synthesized peptide fragments were calculated by a WWW-accessible program ProtParm (http://us.expasy.org/tools/ protparam.html).
3. Results Fig. 1A shows the renal cortical accumulation of [3H]gentamicin 2 h after the intravenous injection with [3H]gentamicin (85 Ag/kg) alone or in combination with megalin substrates such as lysozyme, aprotinin and cytochrome c. Each substrate of megalin used in the present study tended to decrease the accumulation of [3H]gentamicin in the renal cortex. Among them, coadministration of cytochrome c at a dose of 20 mg per rat significantly inhibited the renal accumulation of [3H]gentamicin to the same extent with that by excess amount of unlabeled gentamicin (15 mg per rat). In all cases, there was no significant difference in the plasma concentration-time profile of [3H]gentamicin (1 –120 min), compared with rats injected with [3H]gentamicin alone (data not shown). As shown in Fig. 1B, the combined application of cytochrome c with [3H]gentamicin inhibited the renal accumulation of [3H]gentamicin in a dose-dependent manner. To investigate whether the inhibitory effect of megalin substrates on [3H]gentamicin accumulation in the renal cortex is due to the competitive inhibition of gentamicin uptake via megalin-mediated endocytosis, we examined the effect of cytochrome c on [3H]gentamicin uptake in OK kidney epithelial cells, in which megalin is reportedly expressed [24]. We also confirmed the expression of megalin by Western blotting as well as by ligand blotting with 45Ca2 + in the crude membrane from OK cells used in this study (Fig. 2). Cytochrome c inhibited the time-dependent uptake of [3H]gentamicin in OK cells (Fig. 3A). In addition, cytochrome c showed a concentration-dependent inhibitory effect of [3H]gentamicin uptake in OK cells and its apparent half-maximal inhibitory
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Fig. 2. Detection of megalin in crude membranes from OK cells and rat renal cortex. Immunoblotting (A) and ligand blotting with 45 Ca2 + (B) were performed to confirm the expression of megalin in the OK cells used in the present study. By Western and ligand blotting, megalin was detected in crude membranes of OK cells as well as rat renal cortex.
Fig. 1. Effect of coadministration of various megalin substrates on accumulation of [3H]gentamicin in rat renal cortex. (A) Accumulation of [3H]gentamicin in rat renal cortex was measured at 120 min after the intravenous injection of [3H]gentamicin (25 Ag per rat) without (control) (open column) or with unlabeled gentamicin (15 mg per rat), lysozyme (4.6 mg per rat), aprotinin (2 mg per rat) or cytochrome c (20 mg per rat) (closed columns). (B) [3H]Gentamicin was injected intravenously in combination with various doses of cytochrome c (0.2, 0.6, 2, 20 mg per rat) and the accumulation in renal cortex was measured at 120 min after the injection. Control rats received [3H]gentamicin alone (open circle). Each value is expressed as mean F S.E. of results from three rats. *P < 0.05, significantly different from the value for control.
concentration (IC50) value was 0.94 mM (11.6 mg/ml) (Fig. 3B). The above-mentioned observations suggest that a concomitant administration of megalin substrates may protect gentamicin-induced nephrotoxicity. Therefore, we examined the effect of cytochrome c on gentamicin-induced nephrotoxicity in dehydrated rats, which are more easily affected by aminoglycosides than normal rats. A single dose of gentamicin (30 mg/kg, i.v.) into dehydrated rats significantly increased urinary excretion of NAG, a marker of renal proximal tubular damage, indicating that gentamicin-induced nephrotoxicity was induced, as compared with rats injected the same volume of saline instead of gentamicin (Fig. 4). Furthermore, coadministration of cytochrome c at a dose of 100 mg/kg with gentamicin significantly decreased urinary excretion of NAG compared with rats administered gentamicin alone (Fig. 4). Thus, coadministration of megalin substrates such as cytochrome c may be a useful strategy for preventing aminoglycoside-induced nephrotoxicity. Identification of an active region of megalin substrates, responsible for binding to its receptor, may help to reduce dosage of the compound to prevent aminoglycoside-induced nephrotoxicity. Since basic amino acids have been reported to be essential for the binding of megalin substrates to megalin [10], peptide fragments derived from cytochrome c were designed on the basis of the calculated pI. The syn-
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Cyto70-89 at the concentrations up to 0.5 mM had no significant effect on uptake of [3H]gentamicin in OK cells (data not shown), while Cyto79-88 decreased [3H]gentamicin uptake in OK cells in a concentrationdependent manner with an apparent IC50 value of 0.91 mM ( = 1.1 mg/ml) (Fig. 5). Next, we examined effect of Cyto79-88 under in vivo conditions. The in vivo experiment was performed in mice instead of in rats, because of the limited amounts of the synthetic peptide fragments. Like rats (Fig. 1B), cytochrome c decreased renal accumulation of [3H]gentamicin in mice in a dose-dependent manner (Fig. 6A). On the other hand, Cyto79-88 at a dose of 109 mg/kg had little effect on renal accumulation of [3H]gentamicin in mice (Fig. 6B). The observed differences in inhibitory effect of Cyto79-88 between in OK cells and in mice remain to be clarified, but it may be related to low stability of Cyto79-88 in the body and/or to binding of the peptide fragment to various tissues, which results in decreased concentration in renal tubular lumen.
Fig. 3. Effect of cytochrome c on uptake of [3H]gentamicin in OK kidney epithelial cells. (A) Uptake of [3H]gentamicin (10 AM) without (control, open circles) or with (closed circles) 1 mM cytochrome c was measured at 20, 40 and 60 min. (B) OK cells were incubated with [3H]gentamicin in the absence (control, open circle) or presence (closed circles) of various concentrations of cytochrome c (0.01, 0.05, 0.1, 0.5 and 1 mM). Each value is expressed as mean F S.E. of results from three monolayers. *P < 0.05, significantly different from the value for control.
thesized peptide fragments were Cyto22-41 (KGGKHKTGPNLHGLFGRKTG, pI = 11.33, MW = 2090.4), Cyto70-89 (NPKKYIPGTKMIFAGIKKKG, pI = 10.40, MW = 2219.7) and Cyto79-88 (KMIFAGIKKK, pI = 10.48, MW = 1163.5). Cyto22-41 and
Fig. 4. Effect of coadministration of cytochrome c on urinary excretion of NAG in the dehydrated rats. Dehydrated rats received saline (open column, control), 30 mg/kg gentamicin (closed column, GM) alone or in combination with 100 mg/kg cytochrome c (hatched column, GM + CYT c). Each value is expressed as mean F S.E. of results from four to six rats. *P < 0.05, significantly different from the value for control. yP < 0.05, significantly different from the value for GM.
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(n = 3) or 43.6 F 0.5% of control (n = 3), respectively. In addition, Gelsolin150-169 or CapG137-146 at a dose of 109 mg/kg decreased the renal accumulation of [3H]gentamicin in mice, which was 69.4 F 7.4% of control (n = 4) or 72.5 F 3.8% of control (n = 3), re-
Fig. 5. Effect of Cyto79-88 on uptake of [3H]gentamicin in OK cells. OK cells were incubated with [3H]gentamicin in the absence (control, open column) or presence (closed columns) of various concentrations of Cyto79-88 (0.2, 0.5 and 2 mM). Each value is expressed as mean F S.E. of results from three monolayers. *P < 0.05, significantly different from the value for control.
Furthermore, we investigated effect of peptide fragments derived from actin-regulating proteins, which have been reported to bind phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ). As shown in Fig. 7A, N-WASP181-200 (NISHTKEKKKGKAKKKRLTK, pI = 10.87, MW = 2351.8) inhibited [3H]gentamicin uptake in OK cells. In addition, coadministration of N-WASP181-200 with [3H]gentamicin via the tail vein decreased the renal accumulation of [3H]gentamicin in mice in vivo [Fig. 7B]. Concerning plasma concentration of [3H]gentamicin 120 min after the administration, there was no difference between mice without and with WASP181-200. Furthermore, effects of other peptide fragments on [3H]gentamicin uptake in OK cells and renal accumulation of [3H]gentamicin in mice were examined. [3H]Gentamicin uptake in OK cells in the presence of Gelsolin150-169 (KHVVPNEVVVQRLFQVKGRR, pI = 11.72, MW = 2388.8) or CapG137-146 (KKLYQVKGKK, pI = 10.30, MW = 1219.5) at the concentration of 2 mM was 43.5 F 8.4% of control
Fig. 6. Effect of coadministration of cytochrome c (A) and Cyto7988 (B) on accumulation of [3H]gentamicin in mouse kidney. (A) [3H]Gentamicin (85 Ag/kg) was injected intravenously in combination with various doses of cytochrome c (0.2, 0.6, 2, 20 mg/kg, closed circles) and the accumulation in the kidney was measured at 120 min after the injection. Control mice received [3H]gentamicin alone (open circle). (B) Accumulation of [3H]gentamicin in mouse kidney was measured at 120 min after the intravenous injection of [3H]gentamicin (85 Ag/kg) without (control, open column) or with Cyto79-88 (109 mg/kg, closed column). Each value is expressed as mean F S.E. of results from three mice. *P < 0.05, significantly different from the value for control.
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cells and renal accumulation of [3H]gentamicin in mice.
4. Discussion
Fig. 7. Effect of N-WASP181-200 on uptake of [3H]gentamicin in OK cells (A) and on accumulation of [3H]gentamicin in mouse kidney. (B). (A) OK cells were incubated with [3H]gentamicin in the absence (control, open column) or presence (closed columns) of various concentrations of N-WASP181-200 (0.2, 0.5 and 2 mM). Each value is expressed as mean F S.E. of results from three monolayers. (B) [3H]Gentamicin (85 Ag/kg) was injected intravenously in combination with various doses of N-WASP181-200 (0.1, 1, 10, 30 and 100 mg/kg, closed circles) and the accumulation in the kidney was measured at 120 min after the injection. Control mice received [3H]gentamicin alone (open circle). Each value is expressed as mean F S.E. of results from three mice. *P < 0.05, significantly different from the value for control.
spectively. Thus, like N-WASP181-200, Gelsolin150-169 and CapG137-146 also showed inhibitory effects on [3H]gentamicin uptake in OK
In spite of well-known nephrotoxicity and ototoxicity, aminoglycosides have been the most commonly and widely used antibiotics in the clinical in the last 50 years. Its frequent use may be due to their reliable efficacy and low cost. In addition, much more information concerning their pharmacology, toxicity and therapeutic properties are available than other newer antibacterial agents such as third-generation cephalosporins and third-generation fluoroquinolones. So far, prevention of aminoglycoside-induced side effects has been attempted by numerous researchers and their studies have provided many approaches to attenuate the aminoglycoside-induced toxicity [25 –31]. However, no method to prevent the toxicities has been utilized in the clinical yet. Receptor- or adsorptive-mediated endocytosis has been suggested to be responsible for the concentrated accumulation of aminoglycosides in renal proximal tubule, which is directly related to its nephrotoxicity. Therefore, much attention had been focused on identification of the aminoglycoside binding site at the apical membrane of proximal tubular cells. Identification of macromolecule(s) responsible for the binding has been useful for the molecular-targeted prevention of aminoglycoside-induced nephrotoxicity. Previously, acidic phospholipids in renal brush-border membrane have been identified as the aminoglycoside binding receptor under in vitro conditions [6]. Later, megalin, an endocytic receptor abundantly expressed in the apical membrane of renal proximal tubule, has been suggested as a receptor that plays an important role in the renal aminoglycoside accumulation under in vitro and in vivo studies [10 – 12]. Thus, a compound that interacts with these receptors may be useful for the molecular-targeted prevention of aminoglycoside-induced nephrotoxicity. Previously, some reports demonstrated that polyamino acids such as polyasparagine and polyaspartic acid inhibit the binding of [3H]gentamicin to renal brush-border membranes in vitro and decrease gentamicin-induced nephrotoxicity in vivo, suggesting competition for the aminoglycoside receptor [26,
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32,33]. However, polyaspartic acid did not decrease the renal accumulation of gentamicin and amikacin [32,33]. Therefore, the endocytic receptor such as megalin would not be involved in the interaction between aminoglycosides and polyaspartic acid. On one hand, [3H]gentamicin uptake was decreased in the presence of RAP, a substrate of megalin, in perfused rat proximal tubules [10]. This finding shows that megalin may be useful as a target for prevention of aminoglycoside-induced nephrotoxicity. However, it has not been examined so far whether megalin-targeted prevention of nephrotoxicity induced by aminoglycosides is possible or not. In this study, when [3H]gentamicin was simultaneously injected into rats or mice with megalin substrates such as cytochrome c, decrease in renal accumulation of [3H]gentamicin was observed in both species with no change in the plasma concentrationtime profiles of [3H]gentamicin. This observation suggests that coadministered compound may decrease aminoglycoside-induced nephrotoxicity without affecting the efficacy of aminoglycosides. We therefore examined the effect of cytochrome c on gentamicininduced nephrotoxicity by measuring urinary excretion of NAG, a marker of renal proximal tubular injury. To induce acute aminoglycoside-induced nephrotoxicity, gentamicin was injected to dehydrated rats in which a single injection of aminoglycosides can cause renal failure [19,20]. In fact, an injection of gentamicin at a dose of 30 mg/kg into dehydrated rats increased urinary excretion of NAG. In addition, coadministration of cytochrome c at a dose of 100 mg/kg almost completely attenuated the gentamicin-induced increase in urinary excretion of NAG. We have not examined effect of the coadministration of megalin substrates on gentamicininduced nephrotoxicity in mice because mice have been reported to be much more resistant to aminoglycoside-induced nephrotoxicity than rats [12,34,35]. Taken together, coadministration of megalin substrates with aminoglycoside could be a potent strategy for preventing aminoglycoside-induced nephrotoxicity. On the other hand, to prevent gentamicin-induced nephrotoxicity in the present study, cytochrome c was administrated at a dose of 100 mg/kg into rats. Such a dosage may be an overdose of cytochrome c in the clinical situation. Therefore, to reduce a dose (mg base) of compounds for coadministration, we tested the effect of peptide fragments derived from cyto-
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chrome c on renal accumulation of gentamicin. Since basic amino acids in megalin substrates are reportedly essential for their binding to megalin [10], two peptide fragments with high pI values consisting of 20 amino acids (Cyto22-41, pI = 11.33; Cyto70-89, pI = 10.40) were selected from the whole amino acid sequence of cytochrome c. Furthermore, a 10-amino acid peptide fragment (Cyto79-88, pI = 10.48) was designed from the sequence of Cyto70-89. As a result, one of three peptide fragments (Cyto79-88, pI = 10.40) inhibited uptake of [3H]gentamicin in OK cells in a concentration-dependent manner. The IC50 value of Cyto79-88 on [3H]gentamicin uptake in OK cells (1.1 mg/ml) was lower than that of cytochrome c (11.6 mg/ml) by an order of magnitude. However, unfortunately, [3H]gentamicin accumulation in mouse kidney was not reduced by coadministration of Cyto79-88 at a dose of 109 mg/kg, though the same dose of cytochrome c was sufficient to inhibit [3H]gentamicin accumulation in mouse kidney. No inhibitory effect of Cyto79-88 under in vivo conditions may be due to low stability of Cyto79-88 in the body and/or extensive binding of the peptide fragment to various tissues. Further study will be needed to clarify this point. Interestingly, three peptide fragments derived from actin-regulating proteins such as N-WASP, gelsolin and CapG inhibited not only uptake of [3H]gentamicin in OK cells but also renal accumulation of [3H]gentamicin in mice without affecting the plasma concentration-time profile of [3H]gentamicin. These peptide fragments (N-WASP181-200, Gelsolin150169, CapG137-146) used in this study have been reported to directly bind phosphoinositides such as PIP and PIP2 [16 – 18]. On other hand, the acidic phospholipids have been identified as aminoglycoside receptor at renal brush-border membrane [6]. Therefore, the inhibitory effect of these peptide fragments on the renal [3H]gentamicin accumulation may be due to competitive inhibition for the binding of gentamicin to anionic phospholipids in the apical membrane of renal proximal tubule. The estimated pI values of N-WASP181-200, Gelsolin150-169 and CapG137-146 are 10.87, 11.72 and 10.30, respectively, and cationic at physiological pH. Therefore, these peptides may also bind to megalin and subsequently inhibit the binding and endocytosis of gentamicin via megalin-mediated process. However, the interaction of these peptides with megalin has not been examined
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yet. Further study is necessary to clarify the interaction between these peptides and megalin for the better understanding of the underlying mechanisms. In conclusion, we have found that cationic peptides interacting with megalin and/or acidic phospholipids are very useful for the molecular-targeted prevention of the aminoglycoside-induced nephrotoxicity following reduction of renal accumulation of aminoglycosides. In addition, the present results have suggested that OK cells serve as a valuable model for a rapid screening of candidate compounds to prevent aminoglycoside uptake and toxicity in the kidney. Acknowledgements 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 Uehara Memorial Foundation. We also thank the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University, for the use of their facilities. References [1] G. Kahlmeter, J.I. Dahlager, Aminoglycoside toxicity—a review of clinical studies published between 1975 and 1982, J. Antimicrob. Chemother., (Suppl. A) (1984) 9 – 22. [2] R.L. Slaughter, D.M. Cappelletty, Economic impact of aminoglycoside toxicity and its prevention through therapeutic drug monitoring, Pharmacoeconomics 14 (4) (1998) 385 – 394. [3] B.A. Molitoris, Cell biology of aminoglycoside nephrotoxicity: newer aspects, Curr. Opin. Nephrol. Hypertens. 6 (4) (1997) 384 – 388. [4] M.P. Mingeot-Leclercq, P.M. Tulkens, Aminoglycosides: nephrotoxicity, Antimicrob. Agents Chemother. 43 (5) (1999) 1003 – 1012. [5] M. Takano, Y. Ohishi, M. Okuda, M. Yasuhara, R. Hori, Transport of gentamicin and fluid-phase endocytosis markers in the LLC-PK1 kidney epithelial cell line, J. Pharmacol. Exp. Ther. 268 (2) (1994) 669 – 674. [6] M. Sastrasinh, T.C. Knauss, J.M. Weinberg, H.D. Humes, Identification of the aminoglycoside binding site in rat renal brush border membranes, J. Pharmacol. Exp. Ther. 222 (2) (1982) 350 – 358. [7] E.I. Christensen, H. Birn, P. Verroust, S.K. Moestrup, Membrane receptors for endocytosis in the renal proximal tubule, Int. Rev. Cytol. 180 (1998) 237 – 284. [8] E.I. Christensen, H. Birn, Megalin and cubilin: multifunctional endocytic receptors, Nat. Rev. Mol. Cell. Biol. 3 (4) (2002) 256 – 266.
[9] A. Saito, S. Pietromonaco, A.K. Loo, M.G. Farquhar, Complete cloning and sequencing of rat gp330/‘‘megalin’’, a distinctive member of the low density lipoprotein receptor gene family, Proc. Natl. Acad. Sci. USA 91 (21) (1994) 9725 – 9729. [10] S.K. Moestrup, S. Cui, H. Vorum, C. Bregengard, S.E. Bjorn, K. Norris, J. Gliemann, E.I. Christensen, Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs, J. Clin. Invest. 96 (3) (1995) 1404 – 1413. [11] J. Nagai, H. Tanaka, N. Nakanishi, T. Murakami, M. Takano, Role of megalin in renal handling of aminoglycosides, Am. J. Physiol. Renal Physiol. 281 (2) (2001) F337 – F344. [12] C. Schmitz, J. Hilpert, C. Jacobsen, C. Boensch, E.I. Christensen, F.C. Luft, T.E. Willnow, Megalin deficiency offers protection from renal aminoglycoside accumulation, J. Biol. Chem. 277 (1) (2002) 618 – 622. [13] E.I. Christensen, J. Gliemann, S.K. Moestrup, Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein, J. Histochem. Cytochem. 40 (10) (1992) 1481 – 1490. [14] R.A. Orlando, K. Rader, F. Authier, H. Yamazaki, B.I. Posner, J.J. Bergeron, M.G. Farquhar, Megalin is an endocytic receptor for insulin, J. Am. Soc. Nephrol. 9 (10) (1998) 1759 – 1766. [15] J. Nagai, T. Katsube, T. Murakami, M. Takano, Effect of gentamicin on pharmacokinetics of lysozyme in rats: interaction between megalin substrates in the kidney, J. Pharm. Pharmacol. 54 (11) (2002) 1491 – 1496. [16] P.A. Janmey, J. Lamb, P.G. Allen, P.T. Matsudaira, Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin, J. Biol. Chem. 267 (17) (1992) 11818 – 11823. [17] F.X. Yu, P.A. Johnston, T.C. Sudhof, H.L. Yin, gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein, Science 250 (4986) (1990) 1413 – 1415. [18] K.E. Prehoda, J.A. Scott, R.D. Mullins, W.A. Lim, Integration of multiple signals through cooperative regulation of the NWASP-Arp2/3 complex, Science 290 (5492) (2000) 801 – 806. [19] K. Furuno, K. Ando, S. Suzuki, K. Hirata, Effect of D-glucarates on basic antibiotic-induced renal damage in rats, J. Antibiot. (Tokyo) 29 (2) (1976) 187 – 194. [20] T. Niizato, T. Koeda, T. Tsuruoka, S. Inouye, T. Niida, Protective effect of D-glucaro-delta-lactam against amino-glycoside-induced nephrotoxicity in rats, J. Antibiot. (Tokyo) 29 (8) (1976) 833 – 840. [21] M. Takano, N. Nakanishi, Y. Kitahara, Y. Sasaki, T. Murakami, J. Nagai, Cisplatin-induced inhibition of receptor-mediated endocytosis of protein in the kidney, Kidney Int. 62 (5) (2002) 1707 – 1717. [22] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein – dye binding, Anal. Biochem. 72 (1976) 248 – 254. [23] Y. Sasaki, J. Nagai, Y. Kitahara, N. Takai, T. Murakami, M. Takano, Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells, Biochem. Biophys. Res. Commun. 282 (1) (2001) 212 – 218. [24] X.Y. Zhai, R. Nielsen, H. Birn, K. Drumm, S. Mildenberger, R. Freudinger, S.K. Moestrup, P.J. Verroust, E.I. Christensen, M. Gekle, Cubilin- and megalin-mediated uptake of albumin
A. Watanabe et al. / Journal of Controlled Release 95 (2004) 423–433
[25]
[26]
[27]
[28]
[29]
in cultured proximal tubule cells of opossum kidney, Kidney Int. 58 (4) (2000) 1523 – 1533. S. Inouye, T. Niizato, U. Takeda, T. Koeda, Protective effect of fosfomycin on the experimental nephrotoxicity induced by dibekacin, J. Pharmacobiodyn. 5 (9) (1982) 659 – 669. C. Josepovitz, E. Pastoriza-Munoz, D. Timmerman, M. Scott, S. Feldman, G.J. Kaloyanides, Inhibition of gentamicin uptake in rat renal cortex in vivo by aminoglycosides and organic polycations, J. Pharmacol. Exp. Ther. 223 (2) (1982) 314 – 321. H.D. Humes, M. Sastrasinh, J.M. Weinberg, Calcium is a competitive inhibitor of gentamicin-renal membrane binding interactions and dietary calcium supplementation protects against gentamicin nephrotoxicity, J. Clin. Invest. 73 (1) (1984) 134 – 147. D. Beauchamp, G. Laurent, L. Grenier, P. Gourde, J. Zanen, J.A. Heuson-Stiennon, M.G. Bergeron, Attenuation of gentamicin-induced nephrotoxicity in rats by fleroxacin, Antimicrob. Agents Chemother. 41 (6) (1997) 1237 – 1245. H. Murakami, K. Yayama, L. Chao, J. Chao, Human kallikrein gene delivery protects against gentamycin-induced nephrotoxicity in rats, Kidney Int. 53 (5) (1998) 1305 – 1313.
433
[30] A.A. Shifow, K.V. Kumar, M.U. Naidu, K.S. Ratnakar, Melatonin, a pineal hormone with antioxidant property, protects against gentamicin-induced nephrotoxicity in rats, Nephron 85 (2) (2000) 167 – 174. [31] T. Yazaki, Y. Yoshiyama, P. Wong, D. Beauchamp, M. Kanke, Protective effect of fleroxacin against the nephrotoxicity of isepamicin in rats, Biol. Pharm. Bull. 25 (4) (2002) 516 – 519. [32] P.D. Williams, G.H. Hottendorf, D.B. Bennett, Inhibition of renal membrane binding and nephrotoxicity of aminoglycosides, J. Pharm. Exp. Ther. 237 (3) (1986) 919 – 925. [33] L.S. Ramsammy, C. Josepovitz, B.P. Lane, G.J. Kaloyanides, Polyaspartic acid protects against gentamicin nephrotoxicity in the rat, J. Pharm. Exp. Ther. 250 (1) (1989) 149 – 153. [34] S. Suzuki, S. Hatashima, Y. Shinzawa, O. Niwa, R. Tamatani, Toxicity of neomycin on enzyme activities of kidney and duodenal mucosa in vivo: organ specificity and species difference between rats and mice, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 109 (1) (1994) 77 – 92. [35] S. Suzuki, S. Takamura, J. Yoshida, Y. Shinzawa, O. Niwa, R. Tamatani, Comparison of gentamicin nephrotoxicity between rats and mice, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 112 (1) (1995) 15 – 28.
Targeted prevention of renal accumulation and toxicity of gentamicin by aminoglycoside binding receptor antagonists Ayahisa Watanabe, Junya Nagai, Yoshinori Adachi, Takayuki Katsube, Yasumi Kitahara, Teruo Murakami, Mikihisa Takano * Department of Pharmaceutics and Therapeutics, Division of Clinical Pharmaceutical Science, Programs for Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Received 2 September 2003; accepted 9 December 2003
Abstract Receptor-mediated endocytosis plays an important role in accumulation of aminoglycosides in renal proximal tubule. To prevent aminoglycoside-induced nephrotoxicity following concentrated accumulation of gentamicin in the kidney, effect of cationic proteins and their peptide fragments, which could inhibit gentamicin binding to its binding receptor(s), was investigated. Among several substrates for megalin, an endocytic receptor responsible for renal accumulation of aminoglycosides, cytochrome c potently inhibited gentamicin accumulation in renal cortex. Concentration-dependent inhibition by cytochrome c on gentamicin uptake was also observed in OK kidney epithelial cells expressing megalin. In addition, gentamicin-induced increase in urinary excretion of N-acetyl-h-D-glucosaminidase (NAG), a marker of renal tubular damage, was significantly reduced by cytochrome c. We next attempted to find a peptide fragment with lower molecular size showing inhibitory effect on gentamicin uptake. Cyto79-88 inhibited gentamicin uptake in OK cells, but had little effect on renal accumulation of gentamicin in mice in vivo. On one hand, a peptide fragment of neural Wiskott – Aldrich syndrome protein (N-WASP), which interacts with acidic phospholipids like aminoglycosides, inhibited gentamicin accumulation not only in OK cells but also in mouse kidney. These results show that substrates and/or their peptide fragments for aminoglycoside binding receptor such as megalin might be useful for preventing aminoglycoside-induced nephrotoxicity. D 2004 Elsevier B.V. All rights reserved. Keywords: Aminoglycosides; Nephrotoxicity; Receptor-mediated endocytosis; Megalin; Acidic phospholipids
1. Introduction Aminoglycoside antibiotics are widely used for the treatment of gram-negative infections. However, nephrotoxicity remains a common clinical problem and its toxicity is one of dose-limiting factors in the * Corresponding author. Tel.: +81-82-257-5315; fax: +81-82257-5319. E-mail address: [email protected] (M. Takano). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.12.005
use of aminoglycosides. Reportedly, the average frequencies of nephrotoxicity for gentamicin and tobramycin were 14.0% and 12.9%, respectively [1], whereas it has been also reported that the nephrotoxicity rates range from 1.7% to 58% depending on the definition [2]. In any case, aminoglycoside-induced nephrotoxicity appears to be directly related to the concentrated accumulation of aminoglycosides in the renal proximal tubular cells. Therefore, over the past few decades, a considerable number of studies have
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been made on the pathway responsible for aminoglycoside uptake in the renal proximal tubular cells [3– 5]. Most of aminoglycoside injected into the body is excreted into the urine without being metabolized. However, the rest of the injected dose accumulates selectively and abundantly in the renal cortex. Aminoglycoside taken up by the renal proximal tubular cells stays there for a long time, leading to renal damage such as structural change and functional impairment of plasma membrane, mitochondria and lysosome [4]. Consequently, there has been a great interest in the mechanism with which aminoglycosides are absorbed into the renal proximal tubular cells across the brushborder membrane. So far, many results have indicated that aminoglycosides are taken up by receptor-mediated endocytosis following the binding of aminoglycosides to the brush-border membrane. Sastrasinh et al. [6] suggested the acidic phospholipids as a binding site for aminoglycosides in rat renal brush-border membrane. In the paper, they showed that [3H]gentamicin binds in a saturable manner to several phospholipids such as phosphatidylinositol4,5-bisphosphate (PIP2), phosphatidic acid, phosphatidyl inositol, phosphatidylserine and phosphatidylinositol-4-monophosphate (PIP) [6]. More recently, it has been suggested that megalin, an endocytic receptor expressed at the apical membrane of renal proximal tubules [7 –9], plays an important role in binding and endocytosis of aminoglycosides in renal proximal tubular cells [10 –12]. Megalin belonging to the lowdensity lipoprotein (LDL) receptor family has been shown to bind Ca2 +, vitamin binding proteins such as vitamin D-binding protein and retinal-binding protein, lipoproteins such as apolipoprotein E and apolipoprotein H, enzymes such as lipoprotein lipase and lysozyme, cytochrome c, hemoglobin, drugs such as aminoglycosides, polymixin B, aprotinin, and so on [8,13,14]. Moestrup et al. [10] found that receptorassociated protein (RAP), one of common ligands for LDL receptors, inhibited [3H]gentamicin uptake in perfused rat proximal tubules. In addition, we observed that the intravenous administration of gentamicin to rat increased the urinary excretion of endogenous vitamin D-binding protein [11] and fluorescein isothiocyanate-labeled lysozyme [15]. These results indicate an interaction between gentamicin and megalin substrates at the apical membrane of the renal
proximal tubular cells. Therefore, megalin may be a useful target for attenuating aminoglycoside-induced nephrotoxicity. Actin-regulating proteins such as gelsolin, CapG and neural Wiskott – Aldrich syndrome protein (NWASP) have been reported to have a phosphoinositide-binding region which contains several basic amino acids like megalin substrates. Recently, some phosphoinositide-binding sequences in their actinregulating proteins have been identified [16 – 18]. Since phophoinositides such as PIP2 are also suggested to serve as a receptor responsible for aminoglycoside accumulation in the kidney as described above, a peptide fragment derived from actin-regulating protein, as well as that from a substrate of megalin, may be useful for preventing aminoglycoside-induced nephrotoxicity. In the present study, we demonstrate that megalin substrates such as cytochrome c prevent gentamicininduced nephrotoxicity as well as the accumulation of gentamicin in the renal cortex in rats, probably by decreasing the binding of gentamicin to megalin. Furthermore, cationic peptide fragments, which may interact with anionic phospholipids as well as megalin, significantly inhibited the accumulation of gentamicin in OK cells and in mouse kidney.
2. Materials and methods 2.1. Materials [3H]Gentamicin sulfate (7.4 GBq/g) was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). Lysozyme chloride from egg white, aprotinin from bovine lung and gentamicin sulfate were obtained from Nacalai Tesque (Kyoto, Japan). Cytochrome c from bovine heart (MW = 12,327) was purchased from Sigma Chemical (St. Louis, MO, USA). N-Acetyl-h-D-glucosaminidase (NAG) assay kit was from Shionogi and Co. (Osaka, Japan). All other chemicals used for the experiments were of the highest purity available. 2.2. Preparation of peptide fragments Synthetic peptide fragments were produced with the peptide synthesizer (PSSM-8, Shimazu). The peptide
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fragments were synthesized chemically and their amino acid sequences were as follows: Cyto22-41 (KGGKHKTGPNLHGLFGRKTG), Cyto70-89 (NPKKYIPGTKMIFAGIKKKG), Cyto79-88 (KMIFAGIKKK), N-WASP181-200 (NISHTKEKKKGKAKKKKRLTK), Gelsolin150-169 (KHVVPNEVVVQRLFQVKGRR), and CapG137-146 (KKLYQVKGKK). The peptide sequence of the fragments prepared by the synthesizer was confirmed by matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (PerSeptive Biosystems, Tokyo, Japan). 2.3. In vivo uptake study 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, Graduate School of Biomedical Sciences, Hiroshima University. In rat study, male Wistar rats (230 – 290 g) were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg) and the femoral artery and vein were cannulated with polyethylene tubing for blood sampling and drug administration. The animals were placed on a heating pad to maintain body temperature at 37 jC. [3H]Gentamicin (25 Ag per rat) without or with a candidate antagonist was injected into a femoral vein. Blood samples (50 Al) were withdrawn through the femoral artery at the specified time (1, 5, 10, 20, 30, 45, 60, 90 and 120 min). In mouse study, male ddy mice (22 –28 g) were administered [3H]gentamicin (85 Ag/kg) alone or in combination with a candidate antagonist via the tail vein. Blood samples of mice were obtained by cardiac puncture. At 120 min after the administration into rats or mice, kidneys were excised. In the case of rat study, the renal cortex was dissected from the excised kidney by the use of a Stadie-Riggs microtome. In mouse study, the whole kidney was used to examine the accumulation of [3H]gentamicin. The tissues were weighed and homogenized with 0.25 M sucrose. An aliquot of the homogenates (100 Al) was solubilized with 400 Al of NCSII (Amersham Pharmacia Biotech, Buckinghamshire, UK). Subsequently, 5 ml of ACSII (Amersham Pharmacia Biotech) was added and the radioactivity was measured by liquid scintillation counting.
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2.4. Evaluation of nephrotoxicity Male Wistar rats (210 – 270 g) were maintained without water for 48 h to produce dehydrated rats which are much more susceptible to aminoglycosideinduced renal failure as compared with normal rats [19,20]. The dehydrated rats were injected intravenously with saline (control), 30 mg/kg gentamicin alone (GM), or 30 mg/kg gentamicin with 100 mg/ kg cytochrome c (GM + CYT c). Then, the animals were kept further without water for 17 h. Thereafter, the rats were housed in metabolic cages to collect urine for 24 h with free access to water. NAG in the urine was measured spectrophotometrically using a commercially available kit (Shionogi and Co.). 2.5. Cell culture OK cells were cultured in medium 199 (Gibco BRL, Life Technologies, NY, USA) containing 10% fetal bovine serum (Biological Industries, Israel) without antibiotics, in an atmosphere of 5% CO2 – 95% air at 37 jC, and subcultured every 6 or 7 days using 0.02% EDTA and 0.05% trypsin as described previously [21]. OK cells were used between passages 85 and 98. 2.6. In vitro uptake study Uptake of [3H]gentamicin was measured in OK cells attached to the 12-well plates. Briefly, fresh medium was replaced every 2 or 3 days, and the cells were used on the sixth or seventh days after seeding. Experiments were performed in Dulbecco’s phosphate-buffered saline (PBS buffer containing in mM, 137 NaCl, 3 KCl, 8 Na2HPO4, 1.5 KH2PO4, 0.1 CaCl2 and 0.5 MgCl2) supplemented with 5 mM Dglucose (PBS (G) buffer). After removal of the culture medium, each dish was washed and preincubated with PBS (G) buffer. Then, PBS (G) buffer containing [3H]gentamicin in the absence or presence of a candidate antagonist was added to each dish and the cells were incubated at 37 jC for a specified period. At the end of the incubation, the uptake buffer was aspirated and the dishes were rinsed rapidly three times with 1 ml of ice-cold PBS buffer. The cells were scraped with rubber policeman and then were washed by centrifugation at 4 jC for 1 min at 10,000 rpm twice. After the
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supernatant was aspirated, the pellet was solubilized in 0.1 M NaOH, and the amount of substrate taken up by the cells was measured by counting the radioactivity. Protein was determined by the method of Bradford [22] with bovine serum albumin as the standard. 2.7. Western blot analysis and ligand blotting For immunoblot analysis of megalin in OK cells, the crude membrane fraction from OK cells was prepared the sixth day after seeding as described previously [23]. Briefly, after removal of the culture medium, each dish was washed with ice-cold PBS buffer and the cells were collected with rubber policeman. The cell suspension was homogenized for 2 min with an IKA T25 Basic disperser (IKAR LABORTECHNIK, Germany) in an ice-cold buffer (150 mM NaCl, 1 mM, EDTA, 1 mM PMSF with 20 mM Tris, pH 7.4), and was subsequently homogenized with a glass/Teflon Potter homogenizer with 10 strokes at 1000 rpm. The homogenate was centrifuged at 3000 g for 10 min at 4 jC in an Avanti 30 Compact Centrifuge with rotor F0630. The supernatant was centrifuged at 40,000 g for 30 min at 4 jC. The pellet was resuspended in the ice-cold buffer containing 1% Triton X-100, and centrifuged at 14,000 g for 15 min at 4 jC. The supernatant, which contains crude membrane fractions of OK cells, was heated for 5 min at 95 jC in a loading buffer. The crude membrane fractions of rat renal cortex for control sample of megalin detection were prepared as described in the previous paper [21]. These samples were subjected to SDS-polyacrylamide gel electrophoresis with 6% polyacrylamide gels, and the proteins were transferred for 60 min to PVDF membrane at 4 jC. The membrane was blocked in 5% non-fat dry milk in TBS-T overnight at 4 jC. The membranes were washed three times for 10 min in TBS-T, and were incubated with the anti-rat megalin rabbit antiserum (1:5000 dilution) [11]. The membranes were washed three times in TBS-T, and were incubated with the horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2000 dilution), washed three times in TBS-T, and visualized with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Ligand blotting with 45Ca2 + was carried out as described previously [11,13].
2.8. Data analysis Statistical analysis was performed by Student’s t-test, or by the one way analysis of variance (ANOVA) with the Scheffe´ test for post hoc analysis. A difference of P < 0.05 was considered statistically significant. The isoelectric points (pI) of synthesized peptide fragments were calculated by a WWW-accessible program ProtParm (http://us.expasy.org/tools/ protparam.html).
3. Results Fig. 1A shows the renal cortical accumulation of [3H]gentamicin 2 h after the intravenous injection with [3H]gentamicin (85 Ag/kg) alone or in combination with megalin substrates such as lysozyme, aprotinin and cytochrome c. Each substrate of megalin used in the present study tended to decrease the accumulation of [3H]gentamicin in the renal cortex. Among them, coadministration of cytochrome c at a dose of 20 mg per rat significantly inhibited the renal accumulation of [3H]gentamicin to the same extent with that by excess amount of unlabeled gentamicin (15 mg per rat). In all cases, there was no significant difference in the plasma concentration-time profile of [3H]gentamicin (1 –120 min), compared with rats injected with [3H]gentamicin alone (data not shown). As shown in Fig. 1B, the combined application of cytochrome c with [3H]gentamicin inhibited the renal accumulation of [3H]gentamicin in a dose-dependent manner. To investigate whether the inhibitory effect of megalin substrates on [3H]gentamicin accumulation in the renal cortex is due to the competitive inhibition of gentamicin uptake via megalin-mediated endocytosis, we examined the effect of cytochrome c on [3H]gentamicin uptake in OK kidney epithelial cells, in which megalin is reportedly expressed [24]. We also confirmed the expression of megalin by Western blotting as well as by ligand blotting with 45Ca2 + in the crude membrane from OK cells used in this study (Fig. 2). Cytochrome c inhibited the time-dependent uptake of [3H]gentamicin in OK cells (Fig. 3A). In addition, cytochrome c showed a concentration-dependent inhibitory effect of [3H]gentamicin uptake in OK cells and its apparent half-maximal inhibitory
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Fig. 2. Detection of megalin in crude membranes from OK cells and rat renal cortex. Immunoblotting (A) and ligand blotting with 45 Ca2 + (B) were performed to confirm the expression of megalin in the OK cells used in the present study. By Western and ligand blotting, megalin was detected in crude membranes of OK cells as well as rat renal cortex.
Fig. 1. Effect of coadministration of various megalin substrates on accumulation of [3H]gentamicin in rat renal cortex. (A) Accumulation of [3H]gentamicin in rat renal cortex was measured at 120 min after the intravenous injection of [3H]gentamicin (25 Ag per rat) without (control) (open column) or with unlabeled gentamicin (15 mg per rat), lysozyme (4.6 mg per rat), aprotinin (2 mg per rat) or cytochrome c (20 mg per rat) (closed columns). (B) [3H]Gentamicin was injected intravenously in combination with various doses of cytochrome c (0.2, 0.6, 2, 20 mg per rat) and the accumulation in renal cortex was measured at 120 min after the injection. Control rats received [3H]gentamicin alone (open circle). Each value is expressed as mean F S.E. of results from three rats. *P < 0.05, significantly different from the value for control.
concentration (IC50) value was 0.94 mM (11.6 mg/ml) (Fig. 3B). The above-mentioned observations suggest that a concomitant administration of megalin substrates may protect gentamicin-induced nephrotoxicity. Therefore, we examined the effect of cytochrome c on gentamicin-induced nephrotoxicity in dehydrated rats, which are more easily affected by aminoglycosides than normal rats. A single dose of gentamicin (30 mg/kg, i.v.) into dehydrated rats significantly increased urinary excretion of NAG, a marker of renal proximal tubular damage, indicating that gentamicin-induced nephrotoxicity was induced, as compared with rats injected the same volume of saline instead of gentamicin (Fig. 4). Furthermore, coadministration of cytochrome c at a dose of 100 mg/kg with gentamicin significantly decreased urinary excretion of NAG compared with rats administered gentamicin alone (Fig. 4). Thus, coadministration of megalin substrates such as cytochrome c may be a useful strategy for preventing aminoglycoside-induced nephrotoxicity. Identification of an active region of megalin substrates, responsible for binding to its receptor, may help to reduce dosage of the compound to prevent aminoglycoside-induced nephrotoxicity. Since basic amino acids have been reported to be essential for the binding of megalin substrates to megalin [10], peptide fragments derived from cytochrome c were designed on the basis of the calculated pI. The syn-
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Cyto70-89 at the concentrations up to 0.5 mM had no significant effect on uptake of [3H]gentamicin in OK cells (data not shown), while Cyto79-88 decreased [3H]gentamicin uptake in OK cells in a concentrationdependent manner with an apparent IC50 value of 0.91 mM ( = 1.1 mg/ml) (Fig. 5). Next, we examined effect of Cyto79-88 under in vivo conditions. The in vivo experiment was performed in mice instead of in rats, because of the limited amounts of the synthetic peptide fragments. Like rats (Fig. 1B), cytochrome c decreased renal accumulation of [3H]gentamicin in mice in a dose-dependent manner (Fig. 6A). On the other hand, Cyto79-88 at a dose of 109 mg/kg had little effect on renal accumulation of [3H]gentamicin in mice (Fig. 6B). The observed differences in inhibitory effect of Cyto79-88 between in OK cells and in mice remain to be clarified, but it may be related to low stability of Cyto79-88 in the body and/or to binding of the peptide fragment to various tissues, which results in decreased concentration in renal tubular lumen.
Fig. 3. Effect of cytochrome c on uptake of [3H]gentamicin in OK kidney epithelial cells. (A) Uptake of [3H]gentamicin (10 AM) without (control, open circles) or with (closed circles) 1 mM cytochrome c was measured at 20, 40 and 60 min. (B) OK cells were incubated with [3H]gentamicin in the absence (control, open circle) or presence (closed circles) of various concentrations of cytochrome c (0.01, 0.05, 0.1, 0.5 and 1 mM). Each value is expressed as mean F S.E. of results from three monolayers. *P < 0.05, significantly different from the value for control.
thesized peptide fragments were Cyto22-41 (KGGKHKTGPNLHGLFGRKTG, pI = 11.33, MW = 2090.4), Cyto70-89 (NPKKYIPGTKMIFAGIKKKG, pI = 10.40, MW = 2219.7) and Cyto79-88 (KMIFAGIKKK, pI = 10.48, MW = 1163.5). Cyto22-41 and
Fig. 4. Effect of coadministration of cytochrome c on urinary excretion of NAG in the dehydrated rats. Dehydrated rats received saline (open column, control), 30 mg/kg gentamicin (closed column, GM) alone or in combination with 100 mg/kg cytochrome c (hatched column, GM + CYT c). Each value is expressed as mean F S.E. of results from four to six rats. *P < 0.05, significantly different from the value for control. yP < 0.05, significantly different from the value for GM.
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(n = 3) or 43.6 F 0.5% of control (n = 3), respectively. In addition, Gelsolin150-169 or CapG137-146 at a dose of 109 mg/kg decreased the renal accumulation of [3H]gentamicin in mice, which was 69.4 F 7.4% of control (n = 4) or 72.5 F 3.8% of control (n = 3), re-
Fig. 5. Effect of Cyto79-88 on uptake of [3H]gentamicin in OK cells. OK cells were incubated with [3H]gentamicin in the absence (control, open column) or presence (closed columns) of various concentrations of Cyto79-88 (0.2, 0.5 and 2 mM). Each value is expressed as mean F S.E. of results from three monolayers. *P < 0.05, significantly different from the value for control.
Furthermore, we investigated effect of peptide fragments derived from actin-regulating proteins, which have been reported to bind phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ). As shown in Fig. 7A, N-WASP181-200 (NISHTKEKKKGKAKKKRLTK, pI = 10.87, MW = 2351.8) inhibited [3H]gentamicin uptake in OK cells. In addition, coadministration of N-WASP181-200 with [3H]gentamicin via the tail vein decreased the renal accumulation of [3H]gentamicin in mice in vivo [Fig. 7B]. Concerning plasma concentration of [3H]gentamicin 120 min after the administration, there was no difference between mice without and with WASP181-200. Furthermore, effects of other peptide fragments on [3H]gentamicin uptake in OK cells and renal accumulation of [3H]gentamicin in mice were examined. [3H]Gentamicin uptake in OK cells in the presence of Gelsolin150-169 (KHVVPNEVVVQRLFQVKGRR, pI = 11.72, MW = 2388.8) or CapG137-146 (KKLYQVKGKK, pI = 10.30, MW = 1219.5) at the concentration of 2 mM was 43.5 F 8.4% of control
Fig. 6. Effect of coadministration of cytochrome c (A) and Cyto7988 (B) on accumulation of [3H]gentamicin in mouse kidney. (A) [3H]Gentamicin (85 Ag/kg) was injected intravenously in combination with various doses of cytochrome c (0.2, 0.6, 2, 20 mg/kg, closed circles) and the accumulation in the kidney was measured at 120 min after the injection. Control mice received [3H]gentamicin alone (open circle). (B) Accumulation of [3H]gentamicin in mouse kidney was measured at 120 min after the intravenous injection of [3H]gentamicin (85 Ag/kg) without (control, open column) or with Cyto79-88 (109 mg/kg, closed column). Each value is expressed as mean F S.E. of results from three mice. *P < 0.05, significantly different from the value for control.
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cells and renal accumulation of [3H]gentamicin in mice.
4. Discussion
Fig. 7. Effect of N-WASP181-200 on uptake of [3H]gentamicin in OK cells (A) and on accumulation of [3H]gentamicin in mouse kidney. (B). (A) OK cells were incubated with [3H]gentamicin in the absence (control, open column) or presence (closed columns) of various concentrations of N-WASP181-200 (0.2, 0.5 and 2 mM). Each value is expressed as mean F S.E. of results from three monolayers. (B) [3H]Gentamicin (85 Ag/kg) was injected intravenously in combination with various doses of N-WASP181-200 (0.1, 1, 10, 30 and 100 mg/kg, closed circles) and the accumulation in the kidney was measured at 120 min after the injection. Control mice received [3H]gentamicin alone (open circle). Each value is expressed as mean F S.E. of results from three mice. *P < 0.05, significantly different from the value for control.
spectively. Thus, like N-WASP181-200, Gelsolin150-169 and CapG137-146 also showed inhibitory effects on [3H]gentamicin uptake in OK
In spite of well-known nephrotoxicity and ototoxicity, aminoglycosides have been the most commonly and widely used antibiotics in the clinical in the last 50 years. Its frequent use may be due to their reliable efficacy and low cost. In addition, much more information concerning their pharmacology, toxicity and therapeutic properties are available than other newer antibacterial agents such as third-generation cephalosporins and third-generation fluoroquinolones. So far, prevention of aminoglycoside-induced side effects has been attempted by numerous researchers and their studies have provided many approaches to attenuate the aminoglycoside-induced toxicity [25 –31]. However, no method to prevent the toxicities has been utilized in the clinical yet. Receptor- or adsorptive-mediated endocytosis has been suggested to be responsible for the concentrated accumulation of aminoglycosides in renal proximal tubule, which is directly related to its nephrotoxicity. Therefore, much attention had been focused on identification of the aminoglycoside binding site at the apical membrane of proximal tubular cells. Identification of macromolecule(s) responsible for the binding has been useful for the molecular-targeted prevention of aminoglycoside-induced nephrotoxicity. Previously, acidic phospholipids in renal brush-border membrane have been identified as the aminoglycoside binding receptor under in vitro conditions [6]. Later, megalin, an endocytic receptor abundantly expressed in the apical membrane of renal proximal tubule, has been suggested as a receptor that plays an important role in the renal aminoglycoside accumulation under in vitro and in vivo studies [10 – 12]. Thus, a compound that interacts with these receptors may be useful for the molecular-targeted prevention of aminoglycoside-induced nephrotoxicity. Previously, some reports demonstrated that polyamino acids such as polyasparagine and polyaspartic acid inhibit the binding of [3H]gentamicin to renal brush-border membranes in vitro and decrease gentamicin-induced nephrotoxicity in vivo, suggesting competition for the aminoglycoside receptor [26,
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32,33]. However, polyaspartic acid did not decrease the renal accumulation of gentamicin and amikacin [32,33]. Therefore, the endocytic receptor such as megalin would not be involved in the interaction between aminoglycosides and polyaspartic acid. On one hand, [3H]gentamicin uptake was decreased in the presence of RAP, a substrate of megalin, in perfused rat proximal tubules [10]. This finding shows that megalin may be useful as a target for prevention of aminoglycoside-induced nephrotoxicity. However, it has not been examined so far whether megalin-targeted prevention of nephrotoxicity induced by aminoglycosides is possible or not. In this study, when [3H]gentamicin was simultaneously injected into rats or mice with megalin substrates such as cytochrome c, decrease in renal accumulation of [3H]gentamicin was observed in both species with no change in the plasma concentrationtime profiles of [3H]gentamicin. This observation suggests that coadministered compound may decrease aminoglycoside-induced nephrotoxicity without affecting the efficacy of aminoglycosides. We therefore examined the effect of cytochrome c on gentamicininduced nephrotoxicity by measuring urinary excretion of NAG, a marker of renal proximal tubular injury. To induce acute aminoglycoside-induced nephrotoxicity, gentamicin was injected to dehydrated rats in which a single injection of aminoglycosides can cause renal failure [19,20]. In fact, an injection of gentamicin at a dose of 30 mg/kg into dehydrated rats increased urinary excretion of NAG. In addition, coadministration of cytochrome c at a dose of 100 mg/kg almost completely attenuated the gentamicin-induced increase in urinary excretion of NAG. We have not examined effect of the coadministration of megalin substrates on gentamicininduced nephrotoxicity in mice because mice have been reported to be much more resistant to aminoglycoside-induced nephrotoxicity than rats [12,34,35]. Taken together, coadministration of megalin substrates with aminoglycoside could be a potent strategy for preventing aminoglycoside-induced nephrotoxicity. On the other hand, to prevent gentamicin-induced nephrotoxicity in the present study, cytochrome c was administrated at a dose of 100 mg/kg into rats. Such a dosage may be an overdose of cytochrome c in the clinical situation. Therefore, to reduce a dose (mg base) of compounds for coadministration, we tested the effect of peptide fragments derived from cyto-
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chrome c on renal accumulation of gentamicin. Since basic amino acids in megalin substrates are reportedly essential for their binding to megalin [10], two peptide fragments with high pI values consisting of 20 amino acids (Cyto22-41, pI = 11.33; Cyto70-89, pI = 10.40) were selected from the whole amino acid sequence of cytochrome c. Furthermore, a 10-amino acid peptide fragment (Cyto79-88, pI = 10.48) was designed from the sequence of Cyto70-89. As a result, one of three peptide fragments (Cyto79-88, pI = 10.40) inhibited uptake of [3H]gentamicin in OK cells in a concentration-dependent manner. The IC50 value of Cyto79-88 on [3H]gentamicin uptake in OK cells (1.1 mg/ml) was lower than that of cytochrome c (11.6 mg/ml) by an order of magnitude. However, unfortunately, [3H]gentamicin accumulation in mouse kidney was not reduced by coadministration of Cyto79-88 at a dose of 109 mg/kg, though the same dose of cytochrome c was sufficient to inhibit [3H]gentamicin accumulation in mouse kidney. No inhibitory effect of Cyto79-88 under in vivo conditions may be due to low stability of Cyto79-88 in the body and/or extensive binding of the peptide fragment to various tissues. Further study will be needed to clarify this point. Interestingly, three peptide fragments derived from actin-regulating proteins such as N-WASP, gelsolin and CapG inhibited not only uptake of [3H]gentamicin in OK cells but also renal accumulation of [3H]gentamicin in mice without affecting the plasma concentration-time profile of [3H]gentamicin. These peptide fragments (N-WASP181-200, Gelsolin150169, CapG137-146) used in this study have been reported to directly bind phosphoinositides such as PIP and PIP2 [16 – 18]. On other hand, the acidic phospholipids have been identified as aminoglycoside receptor at renal brush-border membrane [6]. Therefore, the inhibitory effect of these peptide fragments on the renal [3H]gentamicin accumulation may be due to competitive inhibition for the binding of gentamicin to anionic phospholipids in the apical membrane of renal proximal tubule. The estimated pI values of N-WASP181-200, Gelsolin150-169 and CapG137-146 are 10.87, 11.72 and 10.30, respectively, and cationic at physiological pH. Therefore, these peptides may also bind to megalin and subsequently inhibit the binding and endocytosis of gentamicin via megalin-mediated process. However, the interaction of these peptides with megalin has not been examined
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yet. Further study is necessary to clarify the interaction between these peptides and megalin for the better understanding of the underlying mechanisms. In conclusion, we have found that cationic peptides interacting with megalin and/or acidic phospholipids are very useful for the molecular-targeted prevention of the aminoglycoside-induced nephrotoxicity following reduction of renal accumulation of aminoglycosides. In addition, the present results have suggested that OK cells serve as a valuable model for a rapid screening of candidate compounds to prevent aminoglycoside uptake and toxicity in the kidney. Acknowledgements 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 Uehara Memorial Foundation. We also thank the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University, for the use of their facilities. References [1] G. Kahlmeter, J.I. Dahlager, Aminoglycoside toxicity—a review of clinical studies published between 1975 and 1982, J. Antimicrob. Chemother., (Suppl. A) (1984) 9 – 22. [2] R.L. Slaughter, D.M. Cappelletty, Economic impact of aminoglycoside toxicity and its prevention through therapeutic drug monitoring, Pharmacoeconomics 14 (4) (1998) 385 – 394. [3] B.A. Molitoris, Cell biology of aminoglycoside nephrotoxicity: newer aspects, Curr. Opin. Nephrol. Hypertens. 6 (4) (1997) 384 – 388. [4] M.P. Mingeot-Leclercq, P.M. Tulkens, Aminoglycosides: nephrotoxicity, Antimicrob. Agents Chemother. 43 (5) (1999) 1003 – 1012. [5] M. Takano, Y. Ohishi, M. Okuda, M. Yasuhara, R. Hori, Transport of gentamicin and fluid-phase endocytosis markers in the LLC-PK1 kidney epithelial cell line, J. Pharmacol. Exp. Ther. 268 (2) (1994) 669 – 674. [6] M. Sastrasinh, T.C. Knauss, J.M. Weinberg, H.D. Humes, Identification of the aminoglycoside binding site in rat renal brush border membranes, J. Pharmacol. Exp. Ther. 222 (2) (1982) 350 – 358. [7] E.I. Christensen, H. Birn, P. Verroust, S.K. Moestrup, Membrane receptors for endocytosis in the renal proximal tubule, Int. Rev. Cytol. 180 (1998) 237 – 284. [8] E.I. Christensen, H. Birn, Megalin and cubilin: multifunctional endocytic receptors, Nat. Rev. Mol. Cell. Biol. 3 (4) (2002) 256 – 266.
[9] A. Saito, S. Pietromonaco, A.K. Loo, M.G. Farquhar, Complete cloning and sequencing of rat gp330/‘‘megalin’’, a distinctive member of the low density lipoprotein receptor gene family, Proc. Natl. Acad. Sci. USA 91 (21) (1994) 9725 – 9729. [10] S.K. Moestrup, S. Cui, H. Vorum, C. Bregengard, S.E. Bjorn, K. Norris, J. Gliemann, E.I. Christensen, Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs, J. Clin. Invest. 96 (3) (1995) 1404 – 1413. [11] J. Nagai, H. Tanaka, N. Nakanishi, T. Murakami, M. Takano, Role of megalin in renal handling of aminoglycosides, Am. J. Physiol. Renal Physiol. 281 (2) (2001) F337 – F344. [12] C. Schmitz, J. Hilpert, C. Jacobsen, C. Boensch, E.I. Christensen, F.C. Luft, T.E. Willnow, Megalin deficiency offers protection from renal aminoglycoside accumulation, J. Biol. Chem. 277 (1) (2002) 618 – 622. [13] E.I. Christensen, J. Gliemann, S.K. Moestrup, Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein, J. Histochem. Cytochem. 40 (10) (1992) 1481 – 1490. [14] R.A. Orlando, K. Rader, F. Authier, H. Yamazaki, B.I. Posner, J.J. Bergeron, M.G. Farquhar, Megalin is an endocytic receptor for insulin, J. Am. Soc. Nephrol. 9 (10) (1998) 1759 – 1766. [15] J. Nagai, T. Katsube, T. Murakami, M. Takano, Effect of gentamicin on pharmacokinetics of lysozyme in rats: interaction between megalin substrates in the kidney, J. Pharm. Pharmacol. 54 (11) (2002) 1491 – 1496. [16] P.A. Janmey, J. Lamb, P.G. Allen, P.T. Matsudaira, Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin, J. Biol. Chem. 267 (17) (1992) 11818 – 11823. [17] F.X. Yu, P.A. Johnston, T.C. Sudhof, H.L. Yin, gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein, Science 250 (4986) (1990) 1413 – 1415. [18] K.E. Prehoda, J.A. Scott, R.D. Mullins, W.A. Lim, Integration of multiple signals through cooperative regulation of the NWASP-Arp2/3 complex, Science 290 (5492) (2000) 801 – 806. [19] K. Furuno, K. Ando, S. Suzuki, K. Hirata, Effect of D-glucarates on basic antibiotic-induced renal damage in rats, J. Antibiot. (Tokyo) 29 (2) (1976) 187 – 194. [20] T. Niizato, T. Koeda, T. Tsuruoka, S. Inouye, T. Niida, Protective effect of D-glucaro-delta-lactam against amino-glycoside-induced nephrotoxicity in rats, J. Antibiot. (Tokyo) 29 (8) (1976) 833 – 840. [21] M. Takano, N. Nakanishi, Y. Kitahara, Y. Sasaki, T. Murakami, J. Nagai, Cisplatin-induced inhibition of receptor-mediated endocytosis of protein in the kidney, Kidney Int. 62 (5) (2002) 1707 – 1717. [22] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein – dye binding, Anal. Biochem. 72 (1976) 248 – 254. [23] Y. Sasaki, J. Nagai, Y. Kitahara, N. Takai, T. Murakami, M. Takano, Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells, Biochem. Biophys. Res. Commun. 282 (1) (2001) 212 – 218. [24] X.Y. Zhai, R. Nielsen, H. Birn, K. Drumm, S. Mildenberger, R. Freudinger, S.K. Moestrup, P.J. Verroust, E.I. Christensen, M. Gekle, Cubilin- and megalin-mediated uptake of albumin
A. Watanabe et al. / Journal of Controlled Release 95 (2004) 423–433
[25]
[26]
[27]
[28]
[29]
in cultured proximal tubule cells of opossum kidney, Kidney Int. 58 (4) (2000) 1523 – 1533. S. Inouye, T. Niizato, U. Takeda, T. Koeda, Protective effect of fosfomycin on the experimental nephrotoxicity induced by dibekacin, J. Pharmacobiodyn. 5 (9) (1982) 659 – 669. C. Josepovitz, E. Pastoriza-Munoz, D. Timmerman, M. Scott, S. Feldman, G.J. Kaloyanides, Inhibition of gentamicin uptake in rat renal cortex in vivo by aminoglycosides and organic polycations, J. Pharmacol. Exp. Ther. 223 (2) (1982) 314 – 321. H.D. Humes, M. Sastrasinh, J.M. Weinberg, Calcium is a competitive inhibitor of gentamicin-renal membrane binding interactions and dietary calcium supplementation protects against gentamicin nephrotoxicity, J. Clin. Invest. 73 (1) (1984) 134 – 147. D. Beauchamp, G. Laurent, L. Grenier, P. Gourde, J. Zanen, J.A. Heuson-Stiennon, M.G. Bergeron, Attenuation of gentamicin-induced nephrotoxicity in rats by fleroxacin, Antimicrob. Agents Chemother. 41 (6) (1997) 1237 – 1245. H. Murakami, K. Yayama, L. Chao, J. Chao, Human kallikrein gene delivery protects against gentamycin-induced nephrotoxicity in rats, Kidney Int. 53 (5) (1998) 1305 – 1313.
433
[30] A.A. Shifow, K.V. Kumar, M.U. Naidu, K.S. Ratnakar, Melatonin, a pineal hormone with antioxidant property, protects against gentamicin-induced nephrotoxicity in rats, Nephron 85 (2) (2000) 167 – 174. [31] T. Yazaki, Y. Yoshiyama, P. Wong, D. Beauchamp, M. Kanke, Protective effect of fleroxacin against the nephrotoxicity of isepamicin in rats, Biol. Pharm. Bull. 25 (4) (2002) 516 – 519. [32] P.D. Williams, G.H. Hottendorf, D.B. Bennett, Inhibition of renal membrane binding and nephrotoxicity of aminoglycosides, J. Pharm. Exp. Ther. 237 (3) (1986) 919 – 925. [33] L.S. Ramsammy, C. Josepovitz, B.P. Lane, G.J. Kaloyanides, Polyaspartic acid protects against gentamicin nephrotoxicity in the rat, J. Pharm. Exp. Ther. 250 (1) (1989) 149 – 153. [34] S. Suzuki, S. Hatashima, Y. Shinzawa, O. Niwa, R. Tamatani, Toxicity of neomycin on enzyme activities of kidney and duodenal mucosa in vivo: organ specificity and species difference between rats and mice, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 109 (1) (1994) 77 – 92. [35] S. Suzuki, S. Takamura, J. Yoshida, Y. Shinzawa, O. Niwa, R. Tamatani, Comparison of gentamicin nephrotoxicity between rats and mice, Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 112 (1) (1995) 15 – 28.