Tsuneo Deguchi

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: Its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain‐to‐blood transport of indoxyl sulfate at the blood–brain barrier (BBB) using the Brain Efflux Index method. [3H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 × 10−2/min, and the process is saturable with a Km of 298 µm. This process is inhibited by para‐aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acidand 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid. RT–PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [3H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3‐methoxy‐4‐hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6‐mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain‐to‐blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain‐to‐blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.

Rat Organic Anion Transporter 3 (rOAT3) is Responsible for Brain-to-Blood Efflux of Homovanillic Acid at the Abluminal Membrane of Brain Capillary Endothelial Cells

The mechanism that removes homovanillic acid (HVA), an end metabolite of dopamine, from the brain is still poorly understood. The purpose of this study is to identify and characterize the brain-to-blood HVA efflux transporter at the rat blood–brain barrier (BBB). Using the Brain Efflux Index method, the apparent in vivo efflux rate constant of [3H]HVA from the brain, keff, was determined to be 1.69 × 10–2 minute–1. This elimination was significantly inhibited by para-aminohippuric acid (PAH), benzylpenicillin, indoxyl sulfate, and cimetidine, suggesting the involvement of rat organic anion transporter 3 (rOAT3). rOAT3-expressing oocytes exhibited [3H]HVA uptake (Km = 274 μmol/L), which was inhibited by several organic anions, such as PAH, indoxyl sulfate, octanoic acid, and metabolites of monoamine neurotransmitters. Neurotransmitters themselves did not affect the uptake. Furthermore, immunohistochemical analysis suggested that rOAT3 is localized at the abluminal membrane of brain capillary endothelial cells. These results provide the first evidence that rOAT3 is expressed at the abluminal membrane of the rat BBB and is involved in the brain-to-blood transport of HVA. This HVA efflux transport system is likely to play an important role in controlling the level of HVA in the CNS.

Differential Contributions of rOat1 (Slc22a6) and rOat3 (Slc22a8) to the in Vivo Renal Uptake of Uremic Toxins in Rats

No HeadingPurpose.Evidence suggests that uremic toxins such as hippurate (HA), indoleacetate (IA), indoxyl sulfate (IS), and 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) promote the progression of renal failure by damaging tubular cells via rat organic anion transporter 1 (rOat1) and rOat3 on the basolateral membrane of the proximal tubules. The purpose of the current study is to evaluate the in vivo transport mechanism responsible for their renal uptake.Methods.We investigated the uremic toxins transport mechanism using the abdominal aorta injection technique [i.e., kidney uptake index (KUI) method], assuming minimal mixing of the bolus with serum protein from circulating serum.Results.Maximum mixing was estimated to be 5.8% of rat serum by measuring estrone sulfate extraction after addition of 0–90% rat serum to the arterial injection solution. Saturable renal uptake of p-aminohippurate (PAH, Km = 408 μM) and benzylpenicillin (PCG, Km = 346 μM) was observed, respectively. The uptake of PAH and PCG was inhibited in a dose-dependent manner by unlabeled PCG (IC50 = 47.3 mM) and PAH (IC50 = 512 μM), respectively, suggesting that different transporters are responsible for their uptake. A number of uremic toxins inhibited the renal uptake of PAH and PCG. Excess PAH, which could inhibit rOat1 and rOat3, completely inhibited the saturable uptake of IA, IS, and CMPF by the kidney, and by 85% for HA uptake. PCG inhibited the total saturable uptake of HA, IA, IS, and CMPF by 10%, 10%, 45%, and 65%, respectively, at the concentration selective for rOat3.Conclusions.rOat1 could be the primary mediator of the renal uptake of HA and IA, accounting for approximately 75% and 90% of their transport, respectively. rOat1 and rOat3 contributed equally to the renal uptake of IS. rOat3 could account for about 65% of the uptake of CMPF under in vivo physiologic conditions. These results suggest that rOat1 and rOat3 play an important role in the renal uptake of uremic toxins and the induction of their nephrotoxicity.

Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier.

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood–brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [3H]CMPF, [14C]HA and [3H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [3H]CMPF, [14C]HA and [3H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [3H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain‐to‐blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain‐to‐blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.

Human Pharmacokinetic Prediction of UDP-Glucuronosyltransferase Substrates with an Animal Scale-Up Approach

The aim of the current study was to evaluate the accuracy of allometric scaling methods for drugs metabolized by UDP-glucuronosyltransferases (UGTs), such as ketoprofen, imipramine, lorazepam, levofloxacin, zidovudine, diclofenac, furosemide, raloxifene, gemfibrozil, mycophenolic acid, indomethacin, and telmisartan. Human plasma clearance (CL) predictions were conducted from preclinical in vivo data by using multiple-species allometry with the rule of exponents and single-species allometric scaling (SSS) of mice, rats, monkeys, or dogs. Distribution volume at a steady state (Vss) was predicted by multiple-species allometry or SSS of Vss. Oral plasma clearance (CLpo) was calculated under the assumption that Fa × Fg was equivalent across species. Each of the results was compared with the observed parameter calculated from the clinical data after intravenous or oral administration. Multiple-species allometry and SSS of mice, rats, and dogs resulted in a similar accuracy of CL and CLpo predictions. Monkeys tended to provide the most accurate predictions of human CL and CLpo. The ability to predict the half-life, which was determined from CL and Vss predictions, was more accurate in SSS of rats and monkeys. The in vivo fraction metabolized by glucuronidation (fm,UGT) in bile duct-cannulated monkeys was relatively similar to that of humans compared with other animal species, which likely contributed to the highest accuracy of SSS prediction of monkeys. On the basis of the current results, monkeys would be more reliable than other animal species in predicting human pharmacokinetics and fm,UGT for drugs metabolized by UGTs.