Impaired Clearance of Methotrexate in Organic Anion Transporter 3 (Slc22a8) Knockout Mice: A Gender Specific Impact of Reduced Folates

Purpose To elucidate the role of the renal basolateral transporter, Oat3, in the disposition of methotrexate. Materials and Methods Chinese hamster ovary cells expressing mouse Oat3 were used to determine kinetics and specificity of inhibition of methotrexate transport. Methotrexate clearance was then examined in vivo in wildtype and Oat3 knockout mice. Results NSAIDs, ß-lactams, and uremic toxins inhibited mOat3-mediated methotrexate uptake by 70–100%, while folate, leucovorin, and 5-methyltetrahydrofolate inhibited transport by 25–50%. A Km of 60.6±9.3 μM for methotrexate transport was determined. Oat3 knockout mice exhibited reduced methotrexate-to-inulin clearance ratios versus wildtype. Male wildtype mice, but not knockouts or females, demonstrated significantly accelerated methotrexate clearance in response to reduced folates. Reduced folates also markedly inhibited hepatic methotrexate accumulation in males, but not females, and the response was independent of Oat3 function. Conclusions Oat3 contributes to methotrexate clearance, but represents only one component responsible for methotrexate\u27s elimination. Therefore, in patients, dysfunctional hOAT3 polymorphisms or drug competition for hOAT3 transport may severely impact methotrexate elimination only when redundant means of methotrexate removal are also compromised. Furthermore, the present findings suggest that reduced-folate administration only influences methotrexate disposition in males, with the renal reduced-folate response influenced by OAT3 function

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

Analytical Methods for Oxalate Quantification: The Ubiquitous Organic Anion

Oxalate is a divalent organic anion that affects many biological and commercial processes. It is derived from plant sources, such as spinach, rhubarb, tea, cacao, nuts, and beans, and therefore is commonly found in raw or processed food products. Oxalate can also be made endogenously by humans and other mammals as a byproduct of hepatic enzymatic reactions. It is theorized that plants use oxalate to store calcium and protect against herbivory. Clinically, oxalate is best known to be a major component of kidney stones, which commonly contain calcium oxalate crystals. Oxalate can induce an inflammatory response that decreases the immune system’s ability to remove renal crystals. When formulated with platinum as oxaliplatin (an anticancer drug), oxalate has been proposed to cause neurotoxicity and nerve pain. There are many sectors of industry that are hampered by oxalate, and others that depend on it. For example, calcium oxalate is troublesome in the pulp industry and the alumina industry as it deposits on machinery. On the other hand, oxalate is a common active component of rust removal and cleaning products. Due to its ubiquity, there is interest in developing efficient methods to quantify oxalate. Over the past four decades, many diverse methods have been reported. These approaches include electrochemical detection, liquid chromatography or gas chromatography coupled with mass spectrometry, enzymatic degradation of oxalate with oxalate oxidase and detection of hydrogen peroxide produced, and indicator displacement-based methods employing fluorescent or UV light-absorbing compounds. Enhancements in sensitivity have been reported for both electrochemical and mass-spectrometry-based methods as recently as this year. Indicator-based methods have realized a surge in interest that continues to date. The diversity of these approaches, in terms of instrumentation, sample preparation, and sensitivity, has made it clear that no single method will work best for every purpose. This review describes the strengths and limitations of each method, and may serve as a reference for investigators to decide which approach is most suitable for their work

Organic anion transporter 3 (Oat3/Slc22a8) knockout mice exhibit altered clearance and distribution of penicillin G

The interaction of renal basolateral organic anion transporter 3 (Oat3) with commonly used pharmacotherapeutics (e.g., NSAIDs, β-lactams, and methotrexate) has been studied extensively in vitro. However, the in vivo role of Oat3 in drug disposition, in the context of other transporters, glomerular filtration, and metabolism, has not been established. Moreover, recent investigations have identified inactive human OAT3 polymorphisms. Therefore, this investigation was designed to elucidate the in vivo role of Oat3 in the disposition of penicillin G and prototypical substrates using an Oat3 knockout mouse model. Oat3 deletion resulted in a doubling of penicillin’s half-life (P \u3c 0.05) and a reduced volume of distribution (P \u3c 0.01), together yielding a plasma clearance that was one-half (P \u3c 0.05, males) to one-third (P \u3c 0.001, females) of that in wild-type mice. Inhibition of Oat3 abolished the differences in penicillin G elimination between genotypes. Hepatic accumulation of penicillin was 2.3 times higher in male knockouts (P \u3c 0.05) and 3.7 times higher in female knockouts (P \u3c 0.001). Female knockouts also exhibited impaired estrone-3-sulfate clearance. Oat3 deletion did not impact p-aminohippurate elimination, providing correlative evidence to studies in Oat1 knockout mice that suggest Oat1 governs tubular uptake of p-aminohippurate. Collectively, these findings are the first to indicate that functional Oat3 is necessary for proper elimination of xenobiotic and endogenous compounds in vivo. Thus Oat3 plays a distinct role in determining the efficacy and toxicity of drugs. Dysfunctional human OAT3 polymorphisms or instances of polypharmacy involving OAT3 substrates may result in altered systemic accumulation of β-lactams and other clinically relevant compounds

Simultaneous expression of ClopHensor and SLC26A3 reveals the nature of endogenous oxalate transport in CHO cells.

ClopHensor, a fluorescent fusion protein, is a dual function biosensor that has been utilized as a tool for the simultaneous measurement of intracellular chloride and pH in cells. ClopHensor has traditionally been used in conjunction with fluorescence microscopy for single cell measurements. Here, we present a promising multi-well format advancement for the use of ClopHensor as a potential high-throughput method capable of measuring fluorescence signal intensity across a well of confluent cells with highly reproducible results. Using this system, we gained mechanistic insight into an endogenous oxalate transporter in Chinese hamster ovary (CHO) cells expressing ClopHensor and the human chloride transporter, SLC26A3. SLC26A3, a known anion exchanger, has been proposed to play a role in colonic oxalate absorption in humans. Our attempt to study the role of SLC26A3 in oxalate transport revealed the presence of an endogenous oxalate transporter in CHO cells. This transporter was strongly inhibited by niflumate, and exhibited clear saturability. Use of ClopHensor in a multi-well cell assay allowed us to quickly demonstrate that the endogenous oxalate transporter was unable to exchange chloride for bicarbonate, unlike SLC26A3

A Copper(II) Macrocycle Complex for Sensing Biologically Relevant Organic Anions in a Competitive Fluorescence Assay: Oxalate Sensor or Urate Sensor?

Fluorescence sensing of oxalate has garnered some attention in the past two decades as a result of this anion\u27s prominence and impact on society. Previous work on oxalate sensors and other divalent anion sensors has led to the conclusion that the sensors are selective for the anion under investigation. However, sensor selectivity is often determined by testing against a relatively small array of guest molecules or analytes and studies often exclude potentially interfering compounds. For example, studies on oxalate sensors have excluded compounds such as citrate and urate, which are anions in the biological matrices where oxalate is measured