estrone-sulfate and Uremia

The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [

Topics: Biological Transport; Dose-Response Relationship, Drug; Estrone; HEK293 Cells; Hepatocytes; Humans; Indoles; Kinetics; Liver-Specific Organic Anion Transporter 1; Renal Insufficiency; Up-Regulation; Uremia

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.

Topics: Animals; Cell Line; Cell Polarity; Dogs; Estrone; Hydrogen-Ion Concentration; Kidney; Kinetics; Microvilli; Organ Specificity; Organic Anion Transporters; Paraffin Embedding; Protein Transport; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Subcellular Fractions; Tissue Fixation; Uremia

During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [(3)H]-methotrexate ([(3)H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively) and BCRP-mediated [(3)H]-estrone sulfate ([(3)H]-E1S) uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [(3)H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC(50) value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 µM, 4 µM, 129 µM, 1 µM and 18 µM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Cell Membrane; Dose-Response Relationship, Drug; Estrone; Gene Expression Regulation; HEK293 Cells; Humans; Kidney Failure, Chronic; Methotrexate; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Substrate Specificity; Toxins, Biological; Uremia

Chronic kidney disease (CKD) is recognized to cause pharmacokinetic changes in renally excreted drugs; however, pharmacokinetic changes are also reported for drugs that are nonrenally eliminated. Few studies have investigated how uremic toxins may affect drug transporters and metabolizing enzymes and how these may result in pharmacokinetic/metabolic changes in CKD. Here, we investigated the effects of uremic toxins and human uremic serum on the transport of the prototypical transporter substrate [(3) H]-estrone sulfate and three Biopharmaceutics Drug Disposition Classification System (BDDCS) drugs, propranolol, losartan, and eprosartan. We observed a significant decrease in [(3) H]-estrone sulfate, losartan, and eprosartan uptake with some uremic toxins in both transfected cells and rat hepatocytes. The uptake of losartan was decreased in rat and human hepatocytes (28% and 48%, respectively) in the presence of hemodialysis (HD) serum. Time-course studies of losartan showed a 27%, 65%, and 68% increase in area under the curve (AUC) in the presence of HD serum, rifampin, and sulfaphenazole, respectively. Intracellular losartan AUC decreased significantly in the treatment groups, and the metabolite AUC decreased by 41% and 26% in rifampin- and sulfaphenazole-treated group, respectively. The intracellular AUC of eprosartan increased 190% in the presence of HD serum. These studies indicate that the uremic toxins contained in HD serum play an important role in drug disposition through drug transporters, and that there would be differential effects depending on the BDDCS classification of the drug.

Topics: Animals; Area Under Curve; Biopharmaceutics; Chromatography, Liquid; Estrone; Hepatocytes; Humans; Kidney Failure, Chronic; Losartan; Microsomes, Liver; Rats; Tandem Mass Spectrometry; Uremia; Xenobiotics