Genetic variants of human organic anion transporter 4 demonstrate altered transport of endogenous substrates

Abstract

Apical reabsorption from the urine has been shown to be important for such processes as the maintenance of critical metabolites in the blood and the excretion of nephrotoxic compounds. The solute carrier (SLC) transporter OAT4 (SLC22A11) is expressed on the apical membrane of renal proximal tubule cells and is known to mediate the transport of a variety of xenobiotic and endogenous organic anions. Functional characterization of genetic variants of apical transporters thought to mediate reabsorption, such as OAT4, may provide insight into the genetic factors influencing the complex pathways involved in drug elimination and metabolite reclamation occurring in the kidney. Naturally occurring genetic variants of OAT4 were identified in public databases and by resequencing DNA samples from 272 individuals comprising 4 distinct ethnic groups. Nine total nonsynonymous variants were identified and functionally assessed using uptake of three radiolabeled substrates. A nonsense variant, R48Stop, and three other variants (R121C, V155G, and V155M) were found at frequencies of at least 2% in an ethnic group specific fashion. The L29P, R48Stop, and H469R variants displayed a complete loss of function, and kinetic analysis identified a reduced V(max) in the common nonsynonymous variants. Plasma membrane levels of OAT4 protein were absent or reduced in the nonfunctional variants, providing a mechanistic reason for the observed loss of function. Characterization of the genetic variants of reabsorptive transporters such as OAT4 is an important step in understanding variability in tubular reabsorption with important implications in innate homeostatic processes and drug disposition.

Fig. 1.

Location of nonsynonymous variants in human organic anion transporter OAT4. A: sites of nonsynonymous variants in the exonic structure of OAT4. Bold indicates variants that occur at a frequency of at least 1% in at least 1 ethnic group. B: location of nonsynonymous variants in OAT4 secondary structural elements based on predictions using MINNOU (http://minnou.cchmc.org/). Red amino acids indicate the positions of OAT4 nonsynonymous variants.

Fig. 2.

Functional characterization of genetic variants of OAT4 using uptake of canonical substrates. OAT4 mediated uptake of estrone sulfate (A), ochratoxin (B), and uric acid (C). Experiments were performed for 2 (estrone sulfate and ochratoxin A) or 4 min (uric acid) at 37°C using 17 nM [³H]estrone sulfate, 200 μM [³H]ochratoxin A, or 400 μM uric acid (100 μM [¹⁴C]uric acid+300 μM unlabeled uric acid). Uptake values are expressed as a percentage of OAT4 reference sequence uptake and were normalized to total protein in each well. Values are means ± SE of 3 independent experiments. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 compared with reference uptake levels using 1-way ANOVA and Dunnett's post hoc test.

Fig. 3.

Saturable uptake of estrone sulfate in HEK-293-Flp cells overexpressing OAT4 and 3 high-frequency nonsynonymous variants. Experiments were performed for 2 min at 37°C using 20 nM [³H]estrone sulfate and unlabeled estrone sulfate for the remainder; uptake values were normalized to total protein and adjusted for the uptake of estrone sulfate in empty vector-transfected cells. Curves were generated using nonlinear regression with automatic removal of outliers in Graphpad Prism 5.1.

Fig. 4.

mRNA expression levels of OAT4 and its variants in stably transfected HEK-293-Flp cells. Total RNA was extracted from each cell line, reverse transcribed to cDNA, and subsequently used to determine mRNA levels of each variant of OAT4 using quantitative real-time PCR using a Taqman assay specific to human OAT4. Expression levels were normalized to endogenous expression of GAPDH and are shown as a percentage of reference OAT4 expression. Values are means ± SE from 2 independent experiments.

Fig. 5.

Levels of plasma membrane protein in HEK-293-Flp cells expressing reference and nonfunctional forms of OAT4. Top: plasma membrane protein was isolated via biotinylation, separated using SDS-PAGE and probed with an antibody specific to OAT4. Bottom: the same membrane was stripped and reprobed with an antibody specific to the α-subunit of the Na⁺-K⁺-ATPase.