Molecular Mechanism of Ochratoxin A Transport in the Kidney

The mycotoxin, ochratoxin A (OTA), is thought to be responsible for Balkan endemic nephropathy. OTA accumulates in several tissues, especially in the kidneys and liver. The excretion of OTA into urine is thought to be mainly by tubular secretion, presumably via the organic anion transport system. Recently, several families of multispecific organic anion transporters have been identified: organic anion transporters (OATs), organic anion-transporting polypeptides (OATPs), oligopeptide transporters (PEPTs), and ATP-binding cassette (ABC) transporters, such as MRP2 and BCRP. These renal transporters mediate the transmembrane transport of OTA and play a pivotal role in the development of OTA-induced nephrotoxicity

The Role of Transporters in the Pharmacokinetics of Orally Administered Drugs

Drug transporters are recognized as key players in the processes of drug absorption, distribution, metabolism, and elimination. The localization of uptake and efflux transporters in organs responsible for drug biotransformation and excretion gives transporter proteins a unique gatekeeper function in controlling drug access to metabolizing enzymes and excretory pathways. This review seeks to discuss the influence intestinal and hepatic drug transporters have on pharmacokinetic parameters, including bioavailability, exposure, clearance, volume of distribution, and half-life, for orally dosed drugs. This review also describes in detail the Biopharmaceutics Drug Disposition Classification System (BDDCS) and explains how many of the effects drug transporters exert on oral drug pharmacokinetic parameters can be predicted by this classification scheme

Organic Anion Transporter 3 (Oat3/Slc22a8) Interacts with Carboxyfluoroquinolones, and Deletion Increases Systemic Exposure to Ciprofloxacin

Carboxyfluoroquinolones, such as ciprofloxacin, are employed for numerous infectious diseases. Renal secretion is a major determinant of their systemic and urinary concentration, but the specific transporters involved are virtually unknown. In vivo studies implicate the organic anion transporter (OAT) family as a pivotal component of carboxyfluoroquinolone renal secretion. Therefore, this study identified the specific renal basolateral OAT(s) involved, thereby highlighting potential sources of carboxyfluoroquinolone-drug interactions and variable efficacy. Two heterologous expression systems, Xenopus laevis oocytes and cell monolayers, were employed to determine the roles of murine and human renal basolateral mOat1/hOAT1 and mOat3/hOAT3. Ciprofloxacin was transported by mOat3 in both systems (Km, 70±6 μM), and demonstrated no interaction with mOat1 or hOAT1. Furthermore, ciprofloxacin, norfloxacin, ofloxacin, and gatifloxacin exhibited concentration-dependent inhibition of transport on mOat3 in cells, with inhibition constants of 198±39, 558±75, 745±165, and 941±232 μM, respectively. Ciprofloxacin and gatifloxacin also inhibited hOAT3. Subsequently, in vivo elimination of ciprofloxacin was assessed in wild-type and Oat3 null mice (Oat3(-/-)). Oat3(-/-) mice exhibited significantly elevated plasma levels of ciprofloxacin at clinically-relevant concentrations (P\u3c0.05, males; P\u3c0.01, females). Oat3(-/-) mice also demonstrated a reduced volume of distribution (27%, P\u3c0.01, males; 14%, P\u3c0.01, females) and increased area under the concentration-time curve (25%, P\u3c0.05, males; 33%, P\u3c0.01, females). Female Oat3(-/-) mice had a 35% (P\u3c0.01) reduction in total clearance of ciprofloxacin relative to wild-type. Additionally, putative ciprofloxacin metabolites were significantly elevated in Oat3(-/-) mice. The present findings indicate that polymorphisms of, and drug interactions on, hOAT3 may influence carboxyfluoroquinolone efficacy, especially in urinary tract infections

Regulation of Renal Organic Anion Transporter 3 (SLC22A8) Expression and Function by the Integrity of Lipid Raft Domains and their Associated Cytoskeleton

Background/Aims: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. Methods: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. Results: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MβCD. Conclusion: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function

Regulation of Renal Organic Anion Transporter 3 (SLC22A8) Expression and Function by the Integrity of Lipid Raft Domains and their Associated Cytoskeleton

Background/Aims: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. Methods: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. Results: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MβCD. Conclusion: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function

Antispasmodic Effect of Asperidine B, a Pyrrolidine Derivative, through Inhibition of L-Type Ca2+ Channel in Rat Ileal Smooth Muscle

Antispasmodic agents are used for modulating gastrointestinal motility. Several compounds isolated from terrestrial plants have antispasmodic properties. This study aimed to explore the inhibitory effect of the pyrrolidine derivative, asperidine B, isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, on spasmodic activity. Isolated rat ileum was set up in an organ bath. The contractile responses of asperidine B (0.3 to 30 µM) on potassium chloride and acetylcholine-induced contractions were recorded. To investigate its antispasmodic mechanism, CaCl2, acetylcholine, Nω-nitro-l-arginine methyl ester (l-NAME), nifedipine, methylene blue and tetraethylammonium chloride (TEA) were tested in the absence or in the presence of asperidine B. Cumulative concentrations of asperidine B reduced the ileal contraction by ~37%. The calcium chloride and acetylcholine-induced ileal contraction was suppressed by asperidine B. The effects of asperidine B combined with nifedipine, atropine or TEA were similar to those treated with nifedipine, atropine or TEA, respectively. In contrast, in the presence of l-NAME and methylene blue, the antispasmodic effect of asperidine B was unaltered. These results suggest that the antispasmodic property of asperidine B is probably due to the blockage of the L-type Ca2+ channel and is associated with K+ channels and muscarinic receptor, possibly by affecting non-selective cation channels and/or releasing intracellular calcium

Impaired insulin signaling affects renal organic anion transporter 3 (Oat3) function in streptozotocin-induced diabetic rats.

Organic anion transporter 3 (Oat3) is a major renal Oats expressed in the basolateral membrane of renal proximal tubule cells. We have recently reported decreases in renal Oat3 function and expression in diabetic rats and these changes were recovered after insulin treatment for four weeks. However, the mechanisms by which insulin restored these changes have not been elucidated. In this study, we hypothesized that insulin signaling mediators might play a crucial role in the regulation of renal Oat3 function. Experimental diabetic rats were induced by a single intraperitoneal injection of streptozotocin (65 mg/kg). One week after injection, animals showing blood glucose above 250 mg/dL were considered to be diabetic and used for the experiment in which insulin-treated diabetic rats were subcutaneously injected daily with insulin for four weeks. Estrone sulfate (ES) uptake into renal cortical slices was examined to reflect the renal Oat3 function. The results showed that pre-incubation with insulin for 30 min (short term) stimulated [3H]ES uptake into the renal cortical slices of normal control rats. In the untreated diabetic rats, pre-incubation with insulin for 30 min failed to stimulate renal Oat3 activity. The unresponsiveness of renal Oat3 activity to insulin in the untreated diabetic rats suggests the impairment of insulin signaling. Indeed, pre-incubation with phosphoinositide 3-kinase (PI3K) and protein kinase C zeta (PKCζ) inhibitors inhibited insulin-stimulated renal Oat3 activity. In addition, the expressions of PI3K, Akt and PKCζ in the renal cortex of diabetic rats were markedly decreased. Prolonged insulin treatment in diabetic rats restored these alterations toward normal levels. Our data suggest that the decreases in both function and expression of renal Oat3 in diabetes are associated with an impairment of renal insulin-induced Akt/PKB activation through PI3K/PKCζ/Akt/PKB signaling pathway