Indoxyl sulfate (IS) is a typical uremic toxin that extensively accumulates in the plasma of patients with seriously impaired renal function. This study s eeks to clarify whether IS exerts a potent modulating effect on the hepatic transport of pravastatin, which is a substrate of both organic anion transporting peptides (OATPs) and multidrug resistance-associated protein (Mrp) 2 in rats. When IS is administered intravenously to the normal rats at a dose of 120 μmol/kg ; plasma IS levels are approximately 600 μM after 2 min and 100 μM after 120 min. In rats with acute renal failure (ARF) induced by cisplatin, the area under the curve (AUC) was more than 2.5-fold greater compared with that in the normal rats, indicating that IS accumulates in ARF rats. Intravenously administered pravastatin almost disappeared from the plasma by 60 min post-administration and approximately 55% of dose was excreted in the bile within 60 min. This result suggested that pravastatin was efficiently taken up from the sinusoid into hepatocytes via rat OATPs on the sinusoidal membrane and preferentially transported in the bile mediated by Mrp2 on the canalicular membrane. IS administered intravenously at a dose of 120 μmol/kg caused neither an increase in plasma pravastatin levels nor a decrease in its biliary excretion. In conclusion, the present results demonstrate that single intravenous administration of IS does not interfere with the hepatic transport of pravastatin directly in vivo, which is at variance with the results of previous in vitro studies.
It is generally understood that under seriously impaired renal function a variety of uremic toxins accumulate in the body at greater plasma levels, with indoxyl sulfate (IS) typical of such uremic toxins [
There has been a good deal of interest in the alteration of nonrenal drug clearance in patients with severe chronic kidney disease (CKD) [
IS and pravastatin sodium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin was obtained from Nichi-Iko Pharm. Co. (Toyama, Japan). All other regents were of the highest grade available.
In this study, principles of good laboratory animal care were followed and animal experimentation was performed in compliance with the Guidelines for the Care and Use of Laboratory Animals in the Health Sciences University of Hokkaido.
IS was dissolved in saline at a concentration of 11.5 mM or 60 mM, and pravastatin was dissolved in a mixture of polyethylene glycol 400 and saline (1:1) at a concentration of 1 mM. Following cannulation with a polyethylene tube into the bile duct, IS and pravastatin were administered into jugular vein. The doses of IS administered were 23 µmol/kg or 120 µmol/kg, and that of pravastatin was 2 µmol/kg. In this experiment, IS was administered 5 min prior to pravastatin administration. Two doses of IS were based on our preliminary studywhich investigated IS levels in CKD and hemodialysis patients. Blood (0.4 mL) was drawn at 2, 5, 10 20, 30, and 60 min after administration and plasma samples were kept at -30˚C until assay. The bile was collected at 30-minute intervals. ARF rats were induced by the intraperitoneal administration of cisplatin (5 mg/kg) and used after 3 days following confirmation of elevated serum creatinine levels.
After deproteinization with methanol, IS and pravastatin levels in the plasma were assayed using a Shimadzu LC-10A HPLC system (Kyoto, Japan) equipped with a Shimadzu SPD-10 UV spectrophotometer. Chromatographic conditions were as follows: column, Cosmosil 5C18-AR (i.d.: 4.6 mm, length: 15 cm, NakalaiTesque Inc., Kyoto, Japan) for IS and Inertsil ODS-3 (i.d.: 4.6 mm, length: 15 cm, GL Science Inc., Tokyo, Japan) for pravastatin; mobile phase, 0.05M KH2PO4/CH3OH (9:1) for IS and 7:6 and 2.5 mM CH3COONH2/CH3CN (7:3) for pravastatin; wave length, 282 nm for IS and 238 nm for pravastatin; column temperature, 40˚C or 50˚C; flow rate, 1 ml/min; and injection volume, 40 µl. IS and pravastatin were reproducibly assayed with a coefficient of variance of less than 5%.
The AUC was calculated using the trapezoidal method. Data are expressed as the mean with SD of 3 to 4 experiments. Statistical analyses were performed using ANOVA, Dunnett test and Student’s t-test, with p < 0.05 considered to be significant.
First, we investigated the time course of plasma IS levels after intravenous administration to rats with normal renal function at two doses. At the first sampling time point (2 min), plasma IS levels were approximately 600 and 200 µM after administration at the doses of 23 µmol/kg and 120 µmol/kg, respectively (
dose of 23 µmol/kg, that at a dose of 120 µmol/kg was more than 100 µM even at 120 min. When IS was administered to the experimentally induced ARF rats at the same doses, plasma IS levels were much greater compared with those obtained in the normal rats (
Next, we evaluated the effect of IS on the clearance of pravastatin from rat plasma after intravenous administration. Pravastatin is partially excreted in the urine after oral administration in humans [
The biliary excretion of pravastatin is shown in
Dose | ||
---|---|---|
23 μmol/kg | 120 µmol/kg | |
Normal rats | 5371.8 ± 410.9 | 24361.2 ± 1246.5 |
ARF rats | 15724.0 ± 3416.9 | 62240.3 ± 934.9 |
The unit for the AUC was μmol・min/ml.
the bile in an unchanged form within 30 min, and the value remained almost the same after 60 min. The manner of biliary excretion was not altered at all in the presence of IS (
The results of this study showed that intravenously administered pravastatin disappeared very quickly from the plasma in normal rats (
Typically, three members of the OATP family (OATP1B1, OATP1B3, and OATP2B1) are present on the sinusoidal membrane of human hepatocytes, and pravastatin is known to be a substrate of these three OATPs [
Interestingly, pravastatin disappeared from the plasma a little faster in the presence of IS (
Based on previous papers [
To date, several papers have proposed two different mechanisms for the alterations in nonrenal drug clearance induced by uremic toxins including IS; one is the direct inhibition of drug-metabolizing enzymes and/or transporters, and the other is based on the decreased expression of proteins and mRNA due to down-regulation [
In conclusion, our present results suggest that single intravenous administration of IS to normal rats causes only a very limited modulating effect on the transport mediated by OATPs and Mrp2 present in hepatocytes.
We declare no conflict of interest.
Suga, H., Ichimura, Y., Otsuka, S., Sugaya, K., Oda, M. and Saitoh, H. (2018) Limited Effect of Intravenously Administered Indoxyl Sulfate, a Uremic Toxin, on the Hepatic Transport of Pravastatin in Normal Rats. Pharmacology & Pharmacy, 9, 270-278. https://doi.org/10.4236/pp.2018.97021