AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, Y. L.
Right arrow Articles by Hasegawa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, Y. L.
Right arrow Articles by Hasegawa, T.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, May 2002, p. 1522-1528, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1522-1528.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Shiga-Like Toxin II Derived from Escherichia coli O157:H7 Modifies Renal Handling of Levofloxacin in Rats

Ying Lan Zhao,1,2 Xiao Bo Cen,2 Masafumi Ito,3 Keiko Yokoyama,4 Kenji Takagi,1 Kiyoyuki Kitaichi,1 Masayuki Nadai,5 Michio Ohta,4 Kenzo Takagi,1 and Takaaki Hasegawa1*

Department of Medical Technology, Nagoya University School of Health Sciences,1 Department of Pathology,3 Department of Bacteriology, Nagoya University School of Medicine,4 Faculty of Pharmacy, Meijo University, Nagoya, Japan,5 National Safety Assessment Center of Traditional Chinese Medicine, First University Hospital, Sichuan University, Chengdu, China2

Received 9 April 2001/ Returned for modification 26 September 2001/ Accepted 31 January 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effect of Shiga-like toxin II (SLT-II) (2 µg/animal), which was derived from Escherichia coli O157:H7, on renal handling of levofloxacin (LVX), a model drug for quinolone antimicrobial agents, was investigated in rats 24 h after intravenous injection. In histopathological examination, acute tubular injury was observed in SLT-II-treated rats, but the glomeruli were not injured. SLT-II significantly increased the steady-state concentration of LVX in plasma to 1.5-fold that of control rats. SLT-II induced significant decreases in the glomerular filtration rate (GFR) and renal clearance (CLR) of LVX. SLT-II slightly, but significantly, increased the unbound fraction and decreased renal plasma flow with no change in the extraction ratio of p-aminohippurate. SLT-II significantly increased concentrations of tumor necrosis factor alpha (TNF-{alpha}) and nitrite and nitrate (NOx) in plasma. The TNF-{alpha} inhibitor pentoxifylline partly, but significantly, inhibited SLT-II-induced decreases in the GFR and CLR of LVX; in contrast, S-methylisothiourea, a selective inhibitor of inducible nitric oxide synthase, did not. Western blotting analysis revealed that SLT-II did not alter the levels of multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein in kidneys 24 h after injection, assuming the lack of involvement of Mrp2 and P-glycoprotein in SLT-II-induced acute renal tubular injury and renal handling of LVX observed 24 h after SLT-II injection. The present study suggests that SLT-II impairs the renal handling of LVX by decreasing GFR and causing decreased renal plasma flow.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is well-known that drug disposition is altered in some disease states, notably those associated with functional changes in the kidney, which plays a crucial role in drug and metabolite excretion. We have extensively studied changes in the renal excretion of organic anion drugs in cases of renal failure and insufficiency associated with various disease states, especially endotoxemia induced by a gram-negative bacterial infection (2, 9, 20-23, 37).

Escherichia coli O157:H7 infection induces colonization of the bowel and production of powerful Shiga-like toxins (SLTs), which are thought to enter the circulation system and to cause injury to target endothelial cells in various organs, such as the renal glomeruli and the gastrointestinal tract. The SLTs can be divided into two major types: SLT type I (SLT-I) and type II (SLT-II) (16, 24). SLT-II is known to induce nonspecific diarrhea, hemorrhagic colitis, and severe hemolytic-uremic syndrome (HUS). In particular, HUS is the most serious complication of E. coli O157:H7 infection and contributes to renal dysfunction and mortality. However, what and how therapy with antimicrobial agents should be done in the treatment of this infection has not yet been clinically clarified. Relevant animal models for E. coli O157:H7 infection are needed to study the physiological and pathological states of E. coli O157:H7 infectious disease in humans because of the difficulties associated with conducting clinical trials with humans. A wide variety of animal species, such as rabbits, dogs, and mice, have been used as models for human E. coli O157:H7 infections (3, 8, 10). For example, it has been reported that SLT-II isolated from E. coli O167:H7 induces colonic mucosal necrosis and hemorrhage, renal tubular necrosis, and lymphoid necrosis in various tissues in mice (4, 25). In one study, rats were used as an animal model of HUS and hemorrhagic colitis by intravenous injection of SLT-I derived from E. coli O157 (19).

New quinolone antimicrobial agents are sometimes used for the treatment of E. coli O157 infection in Japan. Of the new quinolones, levofloxacin (LVX) has been shown to improve overall mortality of mice infected by E. coli O157:H7 (13). LVX is mainly excreted into the urine by active tubular secretion by drug transporters (38). It has been suggested that LVX is transported by P-glycoprotein (14, 39) and the multispecific organic anion transporter multidrug resistance-associated protein 2 (Mrp2) (27, 28, 34). Therefore, SLT-II might modify the renal handling of LVX by inducing histopathological and physiological changes in kidneys and/or by impairing both drug transporters (Mrp2 and P-glycoprotein). However, the roles of SLT-II in kidney function, renal handling of new quinolone antimicrobial agents, and drug transport systems in humans and animals have not yet been elucidated.

We designed a series of experiments to develop guidelines for the safe use of quinolone antimicrobial agents. We examined the effects of SLT-II on the renal handling of LVX as a model drug for quinolone antimicrobial agents that are excreted mainly into urine and the expression of Mrp2 and P-glycoprotein in the kidney.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. LVX was a kind gift from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). S-Methylisothiourea (SMT), pentoxifylline (PTX), and p-aminohippurate (PAH) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Inulin was purchased from Nacalai Tesque (Kyoto, Japan). Grepafloxacin, which was used as an internal standard, was a kind gift from Otsuka Pharmaceutical Co., Ltd. (Osaka, Japan). All other reagents are commercially available and were of analytical grade.

Preparation of SLT-II. The clinically isolated E. coli O157:H7 strain NGY12 was used for the production of SLT-II. This strain does not produce SLT-I. The absence of the slt-I gene was confirmed by PCR with specific primers. The strain was grown in 500 ml of Luria broth (LB) by constant shaking for 12 h at 37°C. The culture supernatant was obtained by centrifugation, and the protein fraction was precipitated with 60% saturated ammonium sulfate at 4°C. The precipitate was collected by centrifugation, dissolved in 2 ml of phosphate-buffered saline (PBS) (pH 7.2), and dialyzed overnight against PBS at 4°C. The dialysate (approximately 2.5 ml) was used as the crude SLT-II preparation. The concentration of SLT-II in the crude preparation was 20 µg/ml, which was determined by using a reverse passive latex agglutination kit (VTEC-RPLA; Denka Seiken Co., Tokyo, Japan).

Animal experiments. Male Wistar rats (Nippon SLC, Hamamatsu, Japan), weighing 290 to 300 g, were used in this study. The rats were housed under controlled environmental conditions (temperature of 23°C ± 1°C and humidity of 55% ± 5%) with a commercial food diet and water freely available to the animals. All animal experiments were performed according to the guidelines of the Nagoya University School of Medicine for the care and use of laboratory animals.

To determine the effect of SLT-II on the concentration-time curve of LVX in plasma, one day before the experiments, rats were anesthetized with sodium pentobarbital (25 mg/kg of body weight) and the right jugular vein of each rat was cannulated with a polyethylene tube for injection of SLT-II or saline. Twenty-four hours after the intravenous injection of SLT-II (2 µg/animal) or saline, the rats received a single intravenous injection of LVX (10 mg/kg). Blood samples were collected at designated intervals after the injection of LVX. Plasma samples obtained by centrifugation were stored at -40°C until analysis.

To elucidate the effect of SLT-II on renal handling of LVX, 24 h after intravenous injection of SLT-II (2 µg/animal) or saline, the rats (under light anesthesia with pentobarbital) were cannulated with polyethylene tubes into the left jugular artery and the urinary bladder for the collection of blood and urine samples, respectively. All the experiments were done by placing the rats under anesthesia with pentobarbital, and the body temperatures of the animals were maintained at 37°C with a heat lamp. The rats received a bolus intravenous injection of LVX and inulin in a loading dose of 1.5 and 25 mg/kg, respectively, followed by a constant-rate infusion, using an infusion pump (PHD 2000; Harvard Company, South Natick, Mass.), of a 4% mannitol solution delivering doses of 291 µg of LVX/h and 20 mg of inulin/h at a rate of 2 ml/h until the end of the study. A steady-state concentration of LVX in plasma was attained 60 min after the infusion was started. Mannitol was used to obtain sufficient and constant urine flow rate. After 60-min infusion, urine was collected in preweighed tubes at 20-min intervals for 60 min throughout the experiment. Blood samples were taken at the midpoints of the urine sample collection periods (70, 90, and 110 min after the infusion was started). Plasma samples were obtained by centrifugation of the blood samples at 1,200 x g for 10 min. The volume of urine samples was measured gravimetrically with the specific gravity assumed to be 1.0. Plasma and urine samples were stored at -40°C until analysis.

To determine the effects of SLT-II on renal plasma flow (RPF) and extraction ratio (ER), rats received a constant-rate infusion of 15 mg of PAH/h at a rate of 2 ml/h until the end of the study. After 60-min infusion, urine was collected at 10-min intervals for 50 min. Blood samples were taken at the midpoints of the urine sample collection periods (65, 75, 85, 95, and 105 min after the infusion was started). After 110-min infusion, blood samples were collected from the renal vein and carotid artery simultaneously.

To clarify the roles of tumor necrosis factor alpha (TNF-{alpha}) and nitric oxide (NO) in the SLT-II-induced decreases in kidney function and renal excretion of LVX, we measured concentration-time data of TNF-{alpha} and NO in plasma in rats after injection of SLT-II. Next, we investigated the protective effect of PTX, a nonselective inhibitor of TNF-{alpha} production and an inducible nitric oxide synthase (iNOS) inhibitor, SMT, on the steady-state renal excretion of LVX. PTX (50 mg/kg) or SMT (10 mg/kg) was administered intravenously to rats treated with SLT-II or saline 30 min earlier. The doses of PTX and SMT given were determined previously (2, 6, 18). Blood and urine samples were collected by the method described above.

Biochemical determinations. Twenty-four hours after an intravenous administration of saline or SLT-II in rats, blood samples were collected by exsanguinations from the abdominal aortas of rats under light anesthesia with pentobarbital (25 mg/kg). Concentrations of creatinine and urea nitrogen in plasma were measured by an automatic analyzer (model 736-40; Hitachi, Tokyo, Japan). Concentrations of albumin in plasma were determined by the bromcresol green method using a commercial kit (Iatron Albumin Kit; Iatron Laboratories, Tokyo, Japan). Concentrations of TNF-{alpha} in plasma were measured with the Biotrak rat TNF-{alpha} enzyme-linked immunosorbent assay system (Amersham International, Little Chalfont, United Kingdom). Concentrations of nitrite and nitrate (NOx) in plasma were measured with a commercial kit (Nitrate/Nitrite Colorimetric Assay Kit; Cayman Chemical, Ann Arbor, Mich.). The quantitative assay for NOx in plasma was performed by the method previously reported (18).

Histopathological examinations. Rats under light anesthesia with pentobarbital (25 mg/kg) were killed by exsanguinations 24 h after intravenous injection of saline or SLT-II. The kidneys were immediately removed, and small pieces of kidney were fixed in 10% formaldehyde in neutral PBS and routinely embedded in paraffin wax for light microscopy. Paraffin sections were treated with hematoxylin and eosin stain and periodic acid-Schiff reagent. A pathologist performed the histopathological examinations.

Protein binding experiments. To estimate the differences in protein binding of LVX in control rats and SLT-II-treated rats, the protein binding experiment was done by equilibrium dialysis using a cellulose membrane (Visking Sheet; Sanplatec Corp., Osaka, Japan) with the molecular weight cutoff set at 10,000 to 20,000. Blood samples were obtained from the abdominal aortas of control rats (injected with saline) and rats that had been injected with SLT-II 24 h earlier (both sets of rats were lightly anesthesized with ethyl ether), and plasma samples were immediately obtained by centrifugation. Four hundred microliters of a PBS solution (pH 7.4) containing 1 µg of LVX per ml was dialyzed against an equal volume of a fresh plasma sample at 37°C for 8 h to attain equilibrium. Concentrations of LVX on both sides of the membrane were measured by high-performance liquid chromatography (HPLC).

Drug analysis. Concentrations of LVX and PAH in plasma and urine were determined by HPLC. Urine samples were diluted in distilled water. The assay for PAH was performed by a modified version of the method reported previously (1). Fifty microliters of each sample and either 200 µl of methanol containing grepafloxacin (0.1 µg/ml) for LVX or 300 µl of acetonitrile containing p-aminobenzoic acid (3 µg/ml) for PAH as internal standards were mixed and centrifuged at 12,000 x g for 5 min. After centrifugation, the supernatant (200 µl) was then dried under a stream of nitrogen gas at 40°C. The residue was reconstituted in the mobile phase and subjected to HPLC. The LC-6A system (Shimadzu, Kyoto, Japan), equipped with a fluorescence detector (model RF-535; Shimadzu) (emission wavelength, 505 nm; excitation wavelength, 330 nm), or an SPD-6A UV spectrophotometric detector (operated at 254 nm), consisting of an LC-6A liquid pump and an SIL-6A autoinjector, was used for HPLC. HPLC was performed with the following equipment and conditions: Cosmocil 5C18 columns (4.6 by 150 mm; Nacalai Tesque); mobile phase for LVX, 20 mM Na2SO4-acetonitrile (80:20 [vol/vol]) solution containing 0.1% H3PO4; mobile phase for PAH, 50 mM NaHPO4 containing 0.5 mM tetrabutyl ammonium hydrogen sulfate-methanol (90:10 [vol/vol]); column temperature (OTC-6A thermometer; Shimadzu), 40°C for LVX and 50°C for PAH; and flow rate, 1.0 ml/min for LVX and 0.6 ml/min for PAH. These assays were shown to be linear for the concentrations studied with a correlation coefficient of 0.999. No interference with the peak of LVX or PAH was observed in any samples. The within-day and between-day coefficients of variation for this assay were less than 8%. Plasma inulin concentration was measured by the standard colorimetric method (7).

Data analysis. For the renal handling experiments of LVX, the renal clearance (CLR) of LVX, inulin, and PAH during each urine sample collection period was calculated by dividing the urinary excretion rate by the steady-state plasma drug concentration (CSS) determined for that collection period. The renal clearance of drug not bound to plasma protein (CLRU) was calculated by dividing CLR by the plasma-unbound fraction (fU). Glomerular filtration rate (GFR) was calculated as inulin clearance. Assuming that the renal tubular reabsorption of LVX is negligible, the tubular secretion clearance of unbound drug (CLS) was calculated as CLS = CLRU - GFR. Each parameter was calculated by using the mean value for three datum points during 60 min.

RPF was calculated as RPF = CLPAH/ER, where CLPAH is the renal clearance of PAH, respectively. The ER of PAH was calculated as ER = (Cin - Cout)/Cin, where Cin and Cout are the concentrations of PAH entering (carotid artery) and leaving (renal vein) the kidney at the same time point.

Western blot analysis. Kidneys were obtained from control rats and rats treated 12, 24, and 48 h earlier with SLT-II. Each kidney was suspended in 1 ml of 10 mM Tris-HCl buffer (pH 8.0) containing complete protease inhibitor, 1.5 µg of aprotinin per ml, and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemicals). The suspension was homogenized with a tight homogenizer (20 strokes up and down) and centrifuged at 2,000 x g for 15 min at 4°C. The supernatant was centrifuged at 30,000 x g for 60 min at 4°C. The pellet was dissolved in Laemmli buffer and incubated at 37°C for 15 min.

The protein concentration in the solution was measured by Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, Calif.) using bovine serum albumin (Sigma Chemicals) as a standard. The protein (40 µg) was separated by electrophoresis on a sodium dodecyl sulfate-8% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked in PBS containing 0.1% Tween 20 and 5% nonfat dry milk, detected by C219 mouse monoclonal antibody to P-glycoprotein (Dako A/S, Glostrup, Denmark) and human monoclonal antibody to Mrp2 (Alexis Biochemicals, San Diego, Calif.). To quantify the relative levels of P-glycoprotein in each gel, the intensity of the stained bands was measured by NIH image (National Institutes of Health, Bethesda, Md.).

Statistical analysis. Results are expressed as the means ± standard deviations for the indicated number of experiments. Statistical comparisons were assessed by one-way analysis of variance. When the F ratios were significant (P < 0.05), Scheffe's post-hoc tests were done and P values of < 0.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As in human patients with HUS, diarrhea presented as the first definite sign of an E. coli O157:H7 infection and was experienced in most rats treated with SLT-II (2 µg/animal), with all rats dying within 3 or 4 days. Obvious biochemical changes were also observed in rats pretreated with SLT-II. Significant increases in plasma creatinine and urea nitrogen levels were observed in rats treated with SLT-II. As shown in Fig. 1, histopathological examination revealed that the kidneys of SLT-II-injected rats had suffered acute tubular injury. The proximal tubular epithelial cells contained cytoplasmic vacuoles and were disrupted, spotty, and irregularly arranged. However, no significant histopathological changes were observed in glomeruli.



View larger version (139K):
[in this window]
[in a new window]
  		FIG. 1. Light micrograph of tubular cells treated with SLT-II. Magnification, x100.

 
Semilogarithmic plots of plasma LVX concentration-time data after intravenous administration (10 mg/kg) in rats treated with SLT-II or not treated with SLT-II are illustrated in Fig. 2. SLT-II treatment significantly delayed the disappearance of LVX from plasma.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Semilogarithmic plot of concentration-time data of LVX in plasma in control (•) and SLT-II-treated ({circ}) rats after a single intravenous administration of LVX (10 mg/kg). Values are shown as means ± standard deviations (error bars) (n = 5). Values that are significantly different from the control values (P < 0.05) are indicated by the letter a.

 
To determine the effect of SLT-II on the renal handling of LVX, we performed in vivo clearance experiments with the continuous infusion method. The parameters for the renal handling of LVX in the control and SLT-II-treated rats are summarized in Table 1. The steady-state concentrations of LVX in plasma (CSS) in SLT-II-treated rats were significantly higher than those in control rats (0.77 ± 0.11 and 0.49 ± 0.08 µg/ml, respectively). The GFR and CLR of LVX dropped to 50% in SLT-II-treated rats (2.84 to 1.43 ml/min and 4.13 to 2.19 ml/min, respectively). SLT-II significantly decreased albumin concentration in plasma (3.62 to 3.15%) and increased the fU of LVX (0.84 to 0.87). To exclude the effect of plasma protein binding on the renal excretion of LVX, renal clearance for unbound drug (CLRU) was calculated. As a result, the value of CLRU for LVX in SLT-II-treated rats significantly decreased by approximately 50%. Then, assuming that the renal tubular reabsorption of LVX is negligible, the net tubular secretion clearance (CLS) of LVX was decreased in SLT-II-treated rats.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effect of SLT-II on renal handling of LVXa

 
As the renal handling experiments showed that SLT-II decreased the GFR and CLS of LVX, we examined the effects of SLT-II on RPF and PAH. As shown in Table 2, SLT-II significantly decreased renal clearance of PAH (9.09 to 6.54 ml/min) and RPF (11.23 to 8.08 ml/min). However, there was no significant difference in the ER for the control and SLT-II-treated rats.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effects of SLT-II on RPF and ER of PAHa

 
To investigate the roles of TNF-{alpha} and NO in SLT-II-induced reduction in the renal excretion of LVX and in the GFR, the protective effects of PTX (a nonselective inhibitor of TNF-{alpha} production) and SMT (an iNOS inhibitor) against SLT-II-induced reduction of the GFR and CLR of LVX were determined. We also measured levels of TNF-{alpha} and NOx in plasma after intravenous injection of SLT-II. Concentration-time curves of TNF-{alpha} and NOx in plasma after injection of SLT-II are shown in Fig. 3. The concentration of TNF-{alpha} peaked at approximately 1.5 h after injection of SLT-II (approximately 1,500 pg/ml), whereas the concentration of NOx in plasma started to increase 4 to 6 h after injection of SLT-II and peaked at approximately 12 h (approximately 800 µM). The inhibitory effects of PTX and SMT against SLT-II-induced decreases in the GFR and CLR of LVX are illustrated in Fig. 4. PTX significantly inhibited the effect of SLT-II on the GFR and CLR of LVX, but the inhibition was incomplete. On the other hand, SMT had no protective effect against SLT-II-induced decreases in the GFR and CLR of LVX.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3. Concentration-time curves of TNF-{alpha} and NOx in plasma after intravenous injection of SLT-II. Values are shown as means ± standard deviations (error bars) (n = 3).

 


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 4. Protective effects of PTX and SMT against SLT-II-induced decreases in the GFR and CLR of LVX. Means ± standard deviations (error bars) are shown (n = 5). Values that are significantly different from those of the control and SLT-II-treated rats (P < 0.05) are indicated by the letters a and b, respectively.

 
We investigated the effects of SLT-II on the levels of Mrp2 protein and P-glycoprotein in kidney tissue by Western blotting. Figure 5 depicts changes in the levels of Mrp2 and P-glycoprotein as a function of SLT-II. As shown in Fig. 5, SLT-II did not alter the protein level of Mrp2 in the kidney for 48 h after injection. The level of P-glycoprotein remained unchanged for 24 h but decreased to 70% of the control level after 48 h.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 5. P-glycoprotein (A) and Mrp2 (B) expression in the kidneys of rats treated with SLT-II or not treated with SLT-II (control) (top) and relative staining intensity for Mrp2 and P-glycoprotein in control and SLT-II-treated rats (bottom). Kidney samples were taken 12, 24, and 48 h after intravenous injection of SLT-II (2 µg/animal). Each bar represents the intensity ratio compared to the value for the control rats and is presented as the mean ± standard deviation (error bar) (n = 3). The value that is significantly different from the control value (P < 0.05) is indicated by the letter a.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
For HUS patients, it has been reported that glomerular endothelial cells are swollen and detached from the glomerular basement membrane and that glomerular capillaries are occluded with microthrombi (26). In this study using rats, SLT-II induced acute tubular epithelial cell injury without glomerular damage. These results are in accordance with findings by Wadolkowski and colleagues (35, 36), who reported that SLT-II-producing E. coli induced acute renal tubular necrosis in mice, but not glomerular damage. Tesh et al. (32) also reported that SLT-II-induced acute renal tubular necrosis was the only histological lesion in mice. It is likely that the rat model is similar to the mouse model, but the pathological lesions in the kidneys differ from those observed in HUS patients. The differences may be caused by tissue-specific differences in the distribution and expression of globotriosylceramide (Gb3), which is a functional receptor for SLTs.

In the present study, a significant delay in the disappearance of LVX from plasma was observed in SLT-II-treated rats, indicating that SLT-II delays the elimination of LVX from the body. Considering that LVX is excreted primarily into urine and that the protein binding of LVX was very weak (approximately 15%), SLT-II might decrease the renal excretion of LVX. The renal handling experiments clearly showed that in SLT-II-treated rats, the GFR and CLR of LVX decreased by approximately 50% from the values for the control rats. The clearance ratio (CLRU/GFR) of LVX in control rats was approximately 1.8, suggesting that LVX is actively secreted into urine by certain drug transporters. The present study also found that SLT-II decreased RPF. These results suggest that the SLT-II-induced decrease in the CLR of LVX is caused by reduction of GFR and/or tubular secretion. However, the renal ER of PAH did not change, despite renal tubular cell damage. The precise cause cannot be explained at this stage, although tubular cell damage may be moderate. On the basis of these findings, it is presumed that SLT-II-induced decreases in the GFR and CLR of LVX may be partially caused by the decrease in RPF.

Organic anion transporter 1 (OAT1), recently isolated from rat kidney (29, 30), may be one of the multispecific organic anion transporters related to the excretion of anion drugs from the kidney. It has been reported that PAH, but not new quinolone antimicrobial agents, are substrates for OAT1 (15, 29) and that PAH had no inhibitory effect on the uptake of LVX in rat renal cortical slices (14). These observations suggest the lack of involvement of the OAT1-mediated transport system to the renal excretion of LVX.

We recently found that a newly developed quinolone antimicrobial agent, grepafloxacin, is excreted into bile by a P-glycoprotein-mediated transport mechanism (39). Ito and colleagues (14) reported that quinidine, a typical substrate for P-glycoprotein, inhibited the uptake of LVX in rat kidney slices. Thus, it is likely that LVX is a substrate for P-glycoprotein. On the other hand, it has been reported that quinolone antimicrobial agents are substrates for the multispecific organic anion transporter Mrp2 (27, 28, 34).

It is generally thought that renal failure causes the suppression of drug transporter function. We investigated whether SLT-II could reduce the levels of Mrp2 protein and P-glycoprotein related to renal excretion of LVX by Western blotting. However, the levels of expression of Mrp2 and P-glycoprotein cannot explain SLT-II-induced decreases in the CLR and CLS of LVX. It has been reported that plasma samples obtained from rats with acute renal failure inhibit P-glycoprotein-mediated transport system (11), suggesting the presence of endogenous P-glycoprotein substrates in plasma in rats with acute renal failure. One possible explanation may be that endogenous substances or putative Mrp2 and P-glycoprotein substrates in the body inhibit the renal excretion of LVX by modulating Mrp2 and P-glycoprotein transporters because renal failure causes the accumulation of such substances in the body (5, 12, 31). On the basis of these observations, it is suggested that a SLT-II-induced decrease in the CLS of LVX is caused, at least in part, by decreasing RPF and accumulating putative Mrp2 and P-glycoprotein substrates in the body. Consequently, we assume that the unchanged levels of Mrp2 protein and P-glycoprotein in the kidney 24 h after injection of SLT-II play a protective function against SLT-II-induced damage to the kidney by serving to excrete endogenous toxic substances into urine. However, further studies are needed to clarify the existence of other renal transporters responsible for LVX excretion.

There is evidence that SLT-I and SLT-II induce expression of proinflammatory cytokines (33) and that the cytokines interleukin 1 and TNF-{alpha} up-regulate the expression of the toxin-binding neutral glycolipid Gb3 receptor in endothelial cells (17), suggesting that cytokines may play a role in SLT-II-induced histopathological lesions. It is also known that TNF-{alpha} increases NO, which causes tissue damage and excess vasodilation, by inducing iNOS. However, the roles of both the cytokine and mediator in the physiological and pathological changes induced by SLT-II have not yet been fully elucidated. Our previous studies have found that hepatic drug-metabolizing enzyme activity was decreased by SLT-II-induced overproduction of NO in plasma and that a selective iNOS inhibitor, SMT, partly protected the decrease by inhibiting the overproduction of NO (unpublished data). However, this study showed that SMT did not protect SLT-II-induced decreases in the GFR and CLR of LVX. Thus, it is unlikely that the expression of iNOS or overproduction of NO plays a key role in SLT-II-induced kidney dysfunction. We recently reported that pretreatment with PTX significantly inhibited endotoxin-induced increases in TNF-{alpha} levels in plasma (2). In the present study, PTX partly ameliorated SLT-II-induced renal failure and reduction of CLR of LVX, suggesting that TNF-{alpha} is weakly related to SLT-II-induced renal failure.

In conclusion, the present study is the first to report that SLT-II decreases the renal excretion of LVX by decreasing GFR and RPF and induces renal tubular necrosis but has no effect on the levels of Mrp2 protein and P-glycoprotein in the kidney 24 h after injection. The levels of Mrp2 protein and P-glycoprotein could not explain decreased renal excretion of LVX by SLT-II. This phenomenon is probably due to accumulation of endogenous toxic substances and/or putative substrates for Mrp2 and P-glycoprotein in plasma and in the kidney. It is also likely that TNF-{alpha} and NO do not play major roles in SLT-II-induced renal tubular damage. However, further studies will be needed to identify the transporters that play crucial roles in the renal handling of quinolone antimicrobial agents.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Technology, Nagoya University School of Health Sciences, 1-1-20 Daikominami, Higashi-ku, Nagoya 461-8673, Japan. Phone: 81-52-719-3008. Fax: 81-52-719-3009. E-mail: hasegawa{at}met.nagoya-u.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Agarwal, R. 1998. Chromatographic estimation of iothalamate and p-aminohippuric acid to measure glomerular filtration rate and effective renal plasma flow in humans. J. Chromatogr. B 705:3-9.
  2. Ando, H., Y. Nishio, K. Ito, A. Nakao, L. Wang, Y. L. Zhao, K. Kitaichi, K. Takagi, and T. Hasegawa. 2001. Effect of endotoxin on P-glycoprotein-mediated biliary and renal excretion of rhodamine 123 in rats. Antimicrob. Agents Chemother. 45:3462-3467.[Abstract/Free Full Text]
  3. Barrett, T. J., M. E. Potter, and I. K. Wachsmuth. 1989. Bacterial endotoxin both enhances and inhibits the toxicity of Shiga-like toxin II in rabbits and mice. Infect. Immun. 57:3434-3437.[Abstract/Free Full Text]
  4. Beery, D. T., M. P. Doyle, and N. A. Higley. 1984. Cytotoxic activity of Escherichia coli O157:H7 culture filtrate on the mouse colon and kidney. Curr. Microbiol. 11:335-342.[CrossRef]
  5. Charuk, J. H. H., and R. A. F. Reithmeier. 1992. Interaction of P-glycoprotein with a hydrophobic component of rat urine. Biochem. Biophys. Res. Commun. 186:796-802.[CrossRef][Medline]
  6. Chen, Y.-L., V. L. Vraux, J.-P. Giroud, and L. Chauvelot-Moachon. 1994. Anti-tumor necrosis factor properties on non-peptide drugs in acute-phase responses. Eur. J. Pharmacol. 271:319-327.[CrossRef][Medline]
  7. Dische, Z., and E. Borenfreund. 1951. A new spectrophotometric method for the detection and determination of keto sugars and trioses. J. Biol. Chem. 192:583-587.[Free Full Text]
  8. Farmer, J. J., M. E. Potter, L. W. Riley, T. J. Barrett, P. A. Blake, C. A. Bopp, M. L. Cohen, A. Kaufmann, G. K. Morris, R. S. Remis, B. M. Thomason, and J. G. Wells. 1983. Animal models to study Escherichia coli O157:H7 isolated from patients with haemorrhagic colitis. Lancet i:702-703.
  9. Hasegawa, T., M. Nadai, L. Wang, Y. Takayama, K. Kato, T. Nabeshima, and N. Kato. 1994. Renal excretion of famotidine and role of adenosine in renal failure induced by bacterial lipopolysaccharide in rats. Drug Metab. Dispos. 22:8-13.[Abstract]
  10. Hertzke, D. M., L. A. Cowan, P. Schoning, and B. W. Fenwick. 1995. Glomerular ultrastructural lesions of idiopathic cutaneous and renal glomerular vasculopathy of greyhounds. Vet. Pathol. 31:451-459.
  11. Huang, Z.-H., T. Murakami, A. Okochi, R. Yumoto, J. Nagai, and M. Takano. 2000. Expression and function of P-glycoprotein in rats with glycerol-induced acute renal failure. Eur. J. Pharmacol. 406:453-460.[CrossRef][Medline]
  12. Ichikawa, M., A. Yoshimura, T. Furukawa, T. Sumizawa, and S. Akiyama. 1990. Modulators of the multidrug transporter, P-glycoprotein, exist in the human plasma. Biochem. Biophys. Res. Commun. 166:74-80.[CrossRef][Medline]
  13. Isogai, E., H. Isogai, S. Hayashi, T. Kubota, K. Kimura, N. Fujii, T. Ohtani, and K. Sato. 2000. Effect of antibiotics, LVFX and fosfomycin, on a mouse model with Escherichia coli O157 infection. Microbiol. Immunol. 44:89-95.[Medline]
  14. Ito, T., I. Yano, S. Matsuda, Y. Hashimoto, and K. Inui. 1999. Distribution characteristics of LVFX and grepafloxacin in rat kidney. Pharm. Res. 16:534-539.[CrossRef][Medline]
  15. Jariyawat, S., T. Sekine, M. Takeda, N. Apiwattanakul, Y. Kanai, S. Sophasan, and H. Endou. 1999. The interaction and transport of ß-lactam antibiotics with the cloned rat renal organic anion transporter 1. J. Pharmacol. Exp. Ther. 290:672-677.[Abstract/Free Full Text]
  16. Karmali, M. A. 1989. Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2:15-18.[Abstract/Free Full Text]
  17. Kaye, S. A., C. B. Louise, B. Boyd, C. A. Lingwood, and T. G. Obrig. 1993. Shiga toxin-associated hemolytic-uremic syndrome: interleukin-1ß enhancement of Shiga toxin cytotoxicity toward human vascular endothelial cells in vitro. Infect. Immun. 61:3886-3891.[Abstract/Free Full Text]
  18. Kitaichi, K., L. Wang, K. Takagi, M. Iwase, E. Shibata, M. Nadai, K. Takagi, and T. Hasegawa. 1999. Decreased antipyrine clearance following endotoxin administration: in vivo evidence of the role of nitric oxide. Antimicrob. Agents Chemother. 43:2697-2701.[Abstract/Free Full Text]
  19. Matsunaga, T., T. Nakajima, M. Sonoda, S. Kawai, J. Kobayashi, I. Inoue, A. Satomi, S. Katayama, A. Hara, S. Hokari, T. Honda, and T. Komada. 1999. Reactive oxygen species as a risk factor in verotoxin-1-exposed rats. Biochem. Biophys. Res. Commun. 260:813-819.[CrossRef][Medline]
  20. Nadai, M., T. Hasegawa, K. Kato, L. Wang, T. Nabeshima, and N. Kato. 1993. Alterations in pharmacokinetics and protein binding behavior of cefazolin in endotoxemic rats. Antimicrob. Agents Chemother. 37:1781-1785.[Abstract/Free Full Text]
  21. Nadai, M., T. Hasegawa, K. Kato, L. Wang, T. Nabeshima, and N. Kato. 1993. The disposition and renal handling of enprofylline in endotoxemic rats by bacterial lipopolysaccharide (LPS). Drug Metab. Dispos. 21:611-616.[Abstract]
  22. Nadai, M., T. Hasegawa, L. Wang, S. Haghgoo, T. Nabeshima, and N. Kato. 1995. Time-dependent changes in the pharmacokinetics and renal excretion of xanthine derivative enprofylline induced by bacterial endotoxin in rats. Biol. Pharm. Bull. 18:1089-1093.[Medline]
  23. Nadai, M., T. Hasegawa, L. Wang, S. Haghgoo, T. Okasaka, T. Nabeshima, and N. Kato. 1996. Alterations in renal uptake kinetics of the xanthine derivative enprofylline in endotoxaemic mice. J. Pharm. Pharmacol. 48:744-748.[Medline]
  24. O'Brien, A. D., and R. K. Holmes. 1987. Shiga and Shiga-like toxins. Microbiol. Rev. 51:206-220.[Free Full Text]
  25. Padhye, V. V., J. T. Beery, F. B. Kittell, and M. P. Doyle. 1987. Colonic hemorrhage produced in mice by a unique Vero cell cytotoxin from an Escherichia coli strain that causes hemorrhagic colitis. J. Infect. Dis. 155:1249-1253.[Medline]
  26. Richardson, S. E., M. A. Karmali, L. E. Becker, and C. R. Smith. 1988. The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 19:1102-1108.[CrossRef][Medline]
  27. Sasabe, H., A. Tsuji, and Y. Sugiyama. 1998. Carrier-mediated mechanism for the biliary excretion of the quinolone antibiotic grepafloxacin and its glucuronide in rats. J. Pharmacol. Exp. Ther. 284:1033-1039.[Abstract/Free Full Text]
  28. Sasabe, H., Y. Kato, T. Terasaki, A. Tsuji, and Y. Sugiyama. 1999. Differences in the hepatobiliary transport of two quinolone antibiotics, grepafloxacin and lomefloxacin, in the rat. Biopharm. Drug Dispos. 20:151-158.[CrossRef][Medline]
  29. Sekine, T., N. Watanabe, M. Hosoyamada, Y. Kanai, and H. Endou. 1997. Expression, cloning, and characterization of a novel multispecific organic anion transporter. J. Biol. Chem. 272:18526-18529.[Abstract/Free Full Text]
  30. Sweet, D. H., N. A. Wolff, and J. B. Pritchard. 1997. Expression, cloning, and characterization of rOAT1. J. Biol. Chem. 272:30088-30095.[Abstract/Free Full Text]
  31. Tanaka, A., Y. Takahashi, M. Mizokuchi, N. Shimada, and H. Koide. 1999. Plasma, urinary, and erythrocyte concentrations of guanidine compounds in patients with chronic renal failure. Renal Fail. 21:499-514.[Medline]
  32. Tesh, V. L., J. A. Burris, J. W. Owens, V. M. Gordon, W. A. Wadolkowski, A. D. O'Brien, and J. E. Samuel. 1993. Comparison of the relative toxicities of Shiga-like toxins type I and II for mice. Infect. Immun. 61:3392-3402.[Abstract/Free Full Text]
  33. Tesh, V. L., B. Ramegowda, and J. E. Samuel. 1994. Purified Shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages. Infect. Immun. 62:5085-5094.[Abstract/Free Full Text]
  34. Van Aubel, R. A. M. H., J. G. P. Peters, R. Masereeuw, C. H. van Os, and F. G. M. Russel. 2000. Multidrug resistance protein Mrp2 mediates ATP-dependent transport of classic renal organic anion p-aminohippurate. Am. J. Physiol. Renal Physiol. 279:F713-F717.[Abstract/Free Full Text]
  35. Wadolkowski, E. A., J. A. Burris, and A. D. O'Brien. 1990. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 58:2438-2445.[Abstract/Free Full Text]
  36. Wadolkowski, E. A., L. M. Sung, J. A. Burris, J. E. Samuel, and A. D. O'Brien. 1990. Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin type II. Infect. Immun. 58:3959-3965.[Abstract/Free Full Text]
  37. Wang, L., T. Hasegawa, N. Nadai, I. Muraoka, T. Nabeshima, and N. Kato. 1993. The effect of lipopolysaccharide on the disposition of xanthines in rats. J. Pharm. Pharmacol. 45:34-38.[Medline]
  38. Yano, I., T. Ito, M. Takano, and K. Inui. 1997. Evaluation of renal tubular secretion and reabsorption of LVFX in rats. Pharm. Res. 14:508-511.[CrossRef][Medline]
  39. Zhao, Y. L., S. H. Cai, L. Wang, K. Kitaichi, Y. Tatsumi, M. Nadai, H. Yoshizumi, K. Takagi, K. Takagi, and T. Hasegawa. 2002. Possible involvement of P-glycoprotein on biliary excretion of grepafloxacin. Clin. Exp. Pharmacol. Physiol. 29:167-172.[CrossRef][Medline]

Antimicrobial Agents and Chemotherapy, May 2002, p. 1522-1528, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1522-1528.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, Y. L.
Right arrow Articles by Hasegawa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, Y. L.
Right arrow Articles by Hasegawa, T.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS