Douglas H. Sweet

Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology.

Our understanding of the mechanisms behind inter- and intra-patient variability in drug response is inadequate. Advances in the cytochrome P450 drug metabolizing enzyme field have been remarkable, but those in the drug transporter field have trailed behind. Currently, however, interest in carrier-mediated disposition of pharmacotherapeutics is on a substantial uprise. This is exemplified by the 2006 FDA guidance statement directed to the pharmaceutical industry. The guidance recommended that industry ascertain whether novel drug entities interact with transporters. This suggestion likely stems from the observation that several novel cloned transporters contribute significantly to the disposition of various approved drugs. Many drugs bear anionic functional groups, and thus interact with organic anion transporters (OATs). Collectively, these transporters are nearly ubiquitously expressed in barrier epithelia. Moreover, several reports indicate that OATs are subject to diverse forms of regulation, much like drug metabolizing enzymes and receptors. Thus, critical to furthering our understanding of patient- and condition-specific responses to pharmacotherapy is the complete characterization of OAT interactions with drugs and regulatory factors. This review provides the reader with a comprehensive account of the function and substrate profile of cloned OATs. In addition, a major focus of this review is on the regulation of OATs including the impact of transcriptional and epigenetic factors, phosphorylation, hormones and gender.

Two CES1 Gene Mutations Lead to Dysfunctional Carboxylesterase 1 Activity in Man: Clinical Significance and Molecular Basis

The human carboxylesterase 1 (CES1) gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. During the course of a study of the pharmacokinetics of the methyl ester racemic psychostimulant methylphenidate, profoundly elevated methylphenidate plasma concentrations, unprecedented distortions in isomer disposition, and increases in hemodynamic measures were observed in a subject of European descent. These observations led to a focused study of the subjects CES1 gene. DNA sequencing detected two coding region single-nucleotide mutations located in exons 4 and 6. The mutation in exon 4 is located in codon 143 and leads to a nonconservative substitution, p.Gly143Glu. A deletion in exon 6 at codon 260 results in a frameshift mutation, p.Asp260fs, altering residues 260-299 before truncating at a premature stop codon. The minor allele frequency of p.Gly143Glu was determined to be 3.7%, 4.3%, 2.0%, and 0% in white, black, Hispanic, and Asian populations, respectively. Of 925 individual DNA samples examined, none carried the p.Asp260fs, indicating it is an extremely rare mutation. In vitro functional studies demonstrated the catalytic functions of both p.Gly143Glu and p.Asp260fs are substantially impaired, resulting in a complete loss of hydrolytic activity toward methylphenidate. When a more sensitive esterase substrate, p-nitrophenyl acetate was utilized, only 21.4% and 0.6% catalytic efficiency (V(max)/K(m)) were determined in p.Gly143Glu and p.Asp260fs, respectively, compared to the wild-type enzyme. These findings indicate that specific CES1 gene variants can lead to clinically significant alterations in pharmacokinetics and drug response of carboxylesterase 1 substrates.

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

Carboxyfluoroquinolones, such as ciprofloxacin, are used for the treatment of 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 used to determine the roles of murine and human renal basolateral mOat1/hOAT1 and mOat3/hOAT3. Ciprofloxacin was transported by mOat3 in both systems (Km value, 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. Thereafter, 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 < 0.05, male mice; P < 0.01, female mice). Oat3(-/-) mice also demonstrated a reduced volume of distribution (27%, P < 0.01, male mice; 14%, P < 0.01, female mice) and increased area under the concentration-time curve (25%, P < 0.05, male mice; 33%, P < 0.01, female mice). Female Oat3(-/-) mice had a 35% (P < 0.01) reduction in total clearance of ciprofloxacin relative to wild type. In addition, 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.

The furan fatty acid metabolite CMPF is elevated in diabetes and induces β cell dysfunction.

Gestational diabetes (GDM) results from failure of the β cells to adapt to increased metabolic demands; however, the cause of GDM and the extremely high rate of progression to type 2 diabetes (T2D) remains unknown. Using metabolomics, we show that the furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is elevated in the plasma of humans with GDM, as well as impaired glucose-tolerant and T2D patients. In mice, diabetic levels of plasma CMPF induced glucose intolerance, impaired glucose-stimulated insulin secretion, and decreased glucose utilization. Mechanistically, we show that CMPF acts directly on the β cell, causing impaired mitochondrial function, decreasing glucose-induced ATP accumulation, and inducing oxidative stress, resulting in dysregulation of key transcription factors and ultimately reduced insulin biosynthesis. Importantly, specifically blocking its transport through OAT3 or antioxidant treatment could prevent CMPF-induced β cell dysfunction. Thus, CMPF provides a link between β cell dysfunction and GDM/T2D that could be targeted therapeutically.

Renal organic anion transporters (SLC22 family): expression, regulation, roles in toxicity, and impact on injury and disease.

Organic solute flux across the basolateral and apical membranes of renal proximal tubule cells is a key process for maintaining systemic homeostasis. It represents an important route for the elimination of metabolic waste products and xenobiotics, as well as for the reclamation of essential compounds. Members of the organic anion transporter (OAT, SLC22) family expressed in proximal tubules comprise one pathway mediating the active renal secretion and reabsorption of organic anions. Many drugs, pesticides, hormones, heavy metal conjugates, components of phytomedicines, and toxins are OAT substrates. Thus, through transporter activity, the kidney can be a target organ for their beneficial or detrimental effects. Detailed knowledge of the OATs expressed in the kidney, their membrane targeting, substrate specificity, and mechanisms of action is essential to understanding organ function and dysfunction. The intracellular processes controlling OAT expression and function, and that can thus modulate kidney transport capacity, are also critical to this understanding. Such knowledge is also providing insight to new areas such as renal transplant research. This review will provide an overview of the OATs for which transport activity has been demonstrated and expression/function in the kidney observed. Examples establishing a role for renal OATs in drug clearance, food/drug–drug interactions, and renal injury and pathology are presented. An update of the current information regarding the regulation of OAT expression is also provided.

The kidney and uremic toxin removal: Glomerulus or tubule?

Chronic kidney disease (CKD) is a condition that affects approximately 10% of the adult population in developed countries. In patients with CKD adequate renal clearance is compromised, resulting in the accumulation of a plethora of uremic solutes. These uremic retention solutes, also known as uremic toxins, are a heterogeneous group of organic compounds, many are too large to be filtered (middle molecules) or are protein-bound. Tubular secretion shifts the binding and allows for active secretion of such solutes. To mediate urinary solute excretion, renal proximal tubules are equipped with a range of transporters that cooperate in basolateral uptake and luminal excretion. These putative uremic toxins are poorly filtered across dialysis membranes because they are protein bound and current dialysis therapy does not correct the full spectrum of uremic toxicity. Residual renal function, which may represent an important contribution of solutes secreted by the proximal tubule rather than unreabsorbed filtrate, is an important predictor of survival of CKD patients. Many of the transporters that mediate the renal excretion of uremic retention solutes were first recognized as mediators of drug trafficking and drug-drug interactions, and a considerable amount of literature concerning the actions of these transporters antedates the recognition of their importance in the proximal renal tubular transport of uremic retention solutes. These transporters include members belonging to the organic cation/anion/zwitterion solute carrier family, such as the organic anion transporters (OAT)1, OAT3, and OATP4C1, and to the adenosine triphosphate binding cassette superfamily of transmembrane transporters, including the multidrug resistance proteins and breast cancer resistance protein. This article draws on this body of information to describe the renal tubular clearance mechanisms for uremic toxins, as well as the intracellular events associated with their accumulation, involving activation of the aryl hydrocarbon receptor, disturbance of mitochondrial functioning, and competition with metabolizing enzymes.

Potential for food–drug interactions by dietary phenolic acids on human organic anion transporters 1 (SLC22A6), 3 (SLC22A8), and 4 (SLC22A11)

Phenolic acids exert beneficial health effects such as anti-oxidant, anti-carcinogenic, and anti-inflammatory activities and show systemic exposure after consumption of common fruits, vegetables, and beverages. However, knowledge regarding which components convey therapeutic benefits and the mechanism(s) by which they cross cell membranes is extremely limited. Therefore, we determined the inhibitory effects of nine food-derived phenolic acids, p-coumaric acid, ferulic acid, gallic acid, gentisic acid, 4-hydroxybenzoic acid, protocatechuic acid, sinapinic acid, syringic acid, and vanillic acid, on human organic anion transporter 1 (hOAT1), hOAT3, and hOAT4. In the present study, inhibition of OAT-mediated transport of prototypical substrates (1 μM) by phenolic acids (100 μM) was examined in stably expressing cell lines. All compounds significantly inhibited hOAT3 transport, while just ferulic, gallic, protocatechuic, sinapinic, and vanillic acid significantly blocked hOAT1 activity. Only sinapinic acid inhibited hOAT4 (~35%). For compounds exhibiting inhibition > ~60%, known clinical plasma concentration levels and plasma protein binding in humans were examined to select compounds to evaluate further with dose-response curves (IC(50) values) and drug-drug interaction (DDI) index determinations. IC(50) values ranged from 1.24 to 18.08 μM for hOAT1 and from 7.35 to 87.36 μM for hOAT3. Maximum DDI indices for gallic and gentisic acid (≫0.1) indicated a very strong potential for DDIs on hOAT1 and/or hOAT3. This study indicates that gallic acid from foods or supplements, or gentisic acid from salicylate-based drug metabolism, may significantly alter the pharmacokinetics (efficacy and toxicity) of concomitant therapeutics that are hOAT1 and/or hOAT3 substrates.

Linkage of Organic Anion Transporter-1 to Metabolic Pathways through Integrated “Omics”-driven Network and Functional Analysis

The main kidney transporter of many commonly prescribed drugs (e.g. penicillins, diuretics, antivirals, methotrexate, and non-steroidal anti-inflammatory drugs) is organic anion transporter-1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471–6478). Targeted metabolomics in knockouts have shown that OAT1 mediates the secretion or reabsorption of many important metabolites, including intermediates in carbohydrate, fatty acid, and amino acid metabolism. This observation raises the possibility that OAT1 helps regulate broader metabolic activities. We therefore examined the potential roles of OAT1 in metabolic pathways using Recon 1, a functionally tested genome-scale reconstruction of human metabolism. A computational approach was used to analyze in vivo metabolomic as well as transcriptomic data from wild-type and OAT1 knock-out animals, resulting in the implication of several metabolic pathways, including the citric acid cycle, polyamine, and fatty acid metabolism. Validation by in vitro and ex vivo analysis using Xenopus oocyte, cell culture, and kidney tissue assays demonstrated interactions between OAT1 and key intermediates in these metabolic pathways, including previously unknown substrates, such as polyamines (e.g. spermine and spermidine). A genome-scale metabolic network reconstruction generated some experimentally supported predictions for metabolic pathways linked to OAT1-related transport. The data support the possibility that the SLC22 and other families of transporters, known to be expressed in many tissues and primarily known for drug and toxin clearance, are integral to a number of endogenous pathways and may be involved in a larger remote sensing and signaling system (Ahn, S. Y., and Nigam, S. K. (2009) Mol. Pharmacol. 76, 481–490, and Wu, W., Dnyanmote, A. V., and Nigam, S. K. (2011) Mol. Pharmacol. 79, 795–805). Drugs may alter metabolism by competing for OAT1 binding of metabolites.

Activation of Protein Kinase Cζ Increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated Transport

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Cζ (PKCζ) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCζ interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCζ, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCζ activity. Both effects were abolished by a PKCζ-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, ρ-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCζ, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCζ also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCζ acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in Vmax and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCζ activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

Physiological and Molecular Characterization of Aristolochic Acid Transport by the Kidney

Consumption of herbal medicines derived from Aristolochia plants is associated with a progressive tubulointerstitial disease known as aristolochic acid (AA) nephropathy. The nephrotoxin produced naturally by these plants is AA-I, a nitrophenanthrene carboxylic acid that selectively targets the proximal tubule. This nephron segment is prone to toxic injury because of its role in secretory elimination of drugs and other xenobiotics. Here, we characterize the handling of AA-I by membrane transporters involved in renal organic anion transport. Uptake assays in heterologous expression systems identified murine organic anion transporters (mOat1, mOat2, and mOat3) as capable of mediating transport of AA-I. Kinetic analyses showed that all three transporters have an affinity for AA-I in the submicromolar range and thus are likely to operate at toxicologically relevant concentrations in vivo. Structure-activity relationships revealed that the carboxyl group is critical for high-affinity interaction of AA-I with mOat1, mOat2, and mOat3, whereas the nitro group is required only by mOat1. Furthermore, the 8-methoxy group, although essential for toxicity, was not requisite for transport. Mouse renal cortical slices avidly accumulated AA-I, achieving slice-to-medium concentration ratios >10. Uptake by slices was sensitive to known mOat1 and mOat3 substrates and the organic anion transport inhibitor probenecid, which also blocked the production of DNA adducts formed with reactive intracellular metabolites of AA-I. Taken together, these findings indicate that OAT family members mediate high-affinity transport of AA-I and may be involved in the site-selective toxicity and renal elimination of this nephrotoxin.