Maya K. Leabman

Transporters involved in the elimination of drugs in the kidney: Organic anion transporters and organic cation transporters

Transporters in the kidney mediate the secretion or reabsorption of many compounds and thereby influence the plasma levels of their substrates. Organic anion transporters and organic cation transporters are two major classes of secretory transporters in the mammalian kidney. During the past decade, significant progress has been made in the cloning, functional expression, and initial characterization of these transporters. To date, five organic cation transporters and nine organic anion transporters have been cloned. In this review, we summarize the available data on organic anion and organic cation transporters, focusing in particular on their molecular characteristics, tissue distribution, and inhibitor and substrate selectivities. Currently we have a good understanding of the inhibitor selectivities for most of these transporters, and with the development of more robust assays, we will soon have a better understanding of their substrate selectivities. Based on the available data, summarized in this review, it appears that many compounds interact with multiple transporters. Furthermore, there appears to be substantial overlap in the selectivities of organic cation transporters, and the same appears true for organic anion transporters. At the present time, it is unclear what the roles of these multiple transporters are in renal drug elimination. With the development of new assays, reagents, and experimental methods, we will soon have a better understanding of the roles of each transporter isoform in the renal elimination of drugs.

Evolutionary conservation predicts function of variants of the human organic cation transporter, OCT1

The organic cation transporter, OCT1, is a major hepatic transporter that mediates the uptake of many organic cations from the blood into the liver where the compounds may be metabolized or secreted into the bile. Because OCT1 interacts with a variety of structurally diverse organic cations, including clinically used drugs as well as toxic substances (e.g., N-methylpyridinium, MPP+), it is an important determinant of systemic exposure to many xenobiotics. To understand the genetic basis of extensive interindividual differences in xenobiotic disposition, we functionally characterized 15 protein-altering variants of the human liver organic cation transporter, OCT1, in Xenopus oocytes. All variants that reduced or eliminated function (OCT1-R61C, OCT1-P341L, OCT1-G220V, OCT1-G401S, and OCT1-G465R) altered evolutionarily conserved amino acid residues. In general, variants with decreased function had amino acid substitutions that resulted in more radical chemical changes (higher Grantham values) and were less evolutionarily favorable (lower blosum62 values) than variants that maintained function. A variant with increased function (OCT1-S14F) changed an amino acid residue such that the human protein matched the consensus of the OCT1 mammalian orthologs. Our results indicate that changes at evolutionarily conserved positions of OCT1 are strong predictors of decreased function and suggest that a combination of evolutionary conservation and chemical change might be a stronger predictor of function.

Natural variation in human membrane transporter genes reveals evolutionary and functional constraints

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (πNS) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum62, SIFT, and evolutionarily conserved/evolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conserved/evolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.

Polymorphisms in a human kidney xenobiotic transporter, OCT2, exhibit altered function

The completion of the Human Genome Project and the development of high-throughput polymorphism identification methods have allowed researchers to carry out full genetic analyses of many clinically relevant genes. However, few studies have combined genetic analysis with in vitro phenotyping to better understand the relationship between genetic variation and protein function. Many transporters in the kidney are thought to play key roles in defense against a variety of foreign substances. The goal of this study was to understand the relationship between variation in a gene encoding a major renal xenobiotic transporter, OCT2, and transporter function. We report a comprehensive genetic analysis and functional characterization of variants of OCT2. Twenty-eight variable sites in the OCT2 gene were identified in a collection of 247 ethnically diverse DNA samples. Eight caused non-synonymous amino acid changes, of which four were present at >/= 1% in an ethnic population. All four of these altered transporter function assayed in Xenopus laevis oocytes. Analysis of nucleotide diversity (pi) revealed a higher prevalence of synonymous (pi = 22.4 x 10-4) versus non-synonymous (pi = 2.1 x 10-4) changes in OCT2 than in other genes. In addition, the non-synonymous sites had a significant tendency to exhibit more skewed allele frequencies (more negative Tajimas D-values) compared to synonymous sites. The population-genetic analysis, together with the functional characterization, suggests that selection has acted against amino acid changes in OCT2. This selection may be due to a necessary role of OCT2 in the renal elimination of endogenous amines or xenobiotics, including environmental toxins, neurotoxic amines and therapeutic drugs.

Effect of genetic variation in the organic cation transporter 2 on the renal elimination of metformin

Objective The goal of this study was to determine the effect of a genetic variant in the organic cation transporter 2 (OCT2), OCT2-808G/T, which results in an amino acid change, A270S, on the pharmacokinetics of the antidiabetic drug, metformin. Methods The uptake of metformin was performed in stably transfected HEK-293 cells expressing the empty vector (MOCK), the reference OCT2-808G, and the variant OCT2-808T. Healthy individuals with known OCT2 genotypes [14 homozygous for the OCT2 reference allele (808G/G) and nine heterozygous for the variant allele (808G/T, *3D)] were recruited to this study. Metformin concentrations in plasma and urine were measured by liquid chromatography–tandem mass spectrometry method. Creatinine levels were also measured in plasma and urine. Pharmacokinetic parameters were evaluated for both the groups. Results We observed that in HEK-293 stably transfected cells, OCT2-808T had a greater capacity to transport metformin than did the reference OCT2. Metformin pharmacokinetics was characterized in 23 healthy volunteers of Caucasian and African-American ancestries. We observed that the renal clearance (CLR) and the net secretion (SrCLR) of metformin were significantly different between the volunteers heterozygous for the variant allele (808G/T), and the volunteers homozygous for the reference allele (808G/G) (P<0.005). Multivariate analysis revealed that OCT2 genotype was a significant predictor of CLR and SrCLR of metformin (P<0.01). Conclusion We conclude that genetic variation in OCT2 plays an important role in the CLR and SrCLR of metformin in healthy volunteers.

Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1)

Objectives The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics. Methods As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q. Results Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at ≥1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R. Conclusions These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Interactions of n-Tetraalkylammonium Compounds and Biguanides with a Human Renal Organic Cation Transporter (hOCT2)

Many clinically used drugs are transported in the liver and kidney by organic cation transporters (OCT) (1). To date, three organic cation transporters in the OCT family have been cloned and characterized (OCT1-3) (2). Of these, OCT1 appears to be an important transporter in the liver and OCT2 appears to be a major transporter in the kidney. With the availability of the cloned transporters, it is now possible to begin investigating their roles in renal and hepatic drug elimination. The human transporters, hOCT1 and hOCT2, share 70% sequence identity, and their predicted secondary structures, based on hydropathy analysis, are essentially the same. This might suggest that paralogous organic cation transporters such as hOCT1 and hOCT2 have similar functional characteristics and are functionally redundant. However, recent chimeric and mutagenesis studies of transporters have shown that changes in even one or two amino acids can dramatically alter specificity (3). Therefore, it is reasonable to propose that these two organic cation transporter homologs serve different functions in vivo. The goal of this study was to compare the substrate and inhibition profiles of hOCT2 and hOCT1 to determine whether these transporters are functionally distinct. Differences in their specificities may provide insights into organ-specific elimination of organic cations. We examined the interactions of n-tetraalkylammonium (nTAA) compounds and biguanides with hOCT2 and compared our results with our previous results for hOCT1 (4,5). Substantial differences between hOCT2 and hOCT1 in their interactions with nTAAs were found, whereas their interactions with the biguanides, metformin and phenformin, were similar. This report demonstrates that there are compound-dependent differences in the specificities of hOCT1 and hOCT2; these differences may contribute to organ-specific elimination of drugs. MATERIALS AND METHODS

Heritability of metformin renal clearance

Twin studies have been used to determine the genetic contribution to variation in hepatic drug metabolism. In contrast, little is known about the contribution of genetic factors to variation in renal drug elimination. In this study, we used monozygotic twin pairs to assess the heritability of the clearance of the anti‐diabetic agent, metformin, a drug that is eliminated exclusively in the kidneys.

SNP analysis and presentation in the Pharmacogenetics of Membrane Transporters Project.

The multidisciplinary UCSF Pharmacogenetics of Membrane Transporters project seeks to systematically identify sequence variants in transporters and to determine the functional significance of these variants through evaluation of relevant cellular and clinical phenotypes. The project is structured around four interacting cores: genomics, cellular phenotyping, clinical phenotyping, and bioinformatics. The bioinformatics core is responsible for collecting, storing, and analyzing the information obtained by the other cores and for presenting the results, in particular, for the genomic data. Most of this process is automated using locally developed software written in Python, an open source language well suited for rapid, modular development that meets requirements that are themselves constantly evolving. Here we present the details of transforming ABI trace file data into useful information for project investigators and a description of the types of data analysis and display that we have developed.