Apical Voltage-Driven Urate Efflux Transporter NPT4 in Renal Proximal Tubule

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

Increased expression of SLC2A9 decreases urate excretion from the kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K(m): 365+/-42 microm). The transport was Na(+)-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; urate [corrected] is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).     

Expression of SLC2A9 Isoforms in the Kidney and Their Localization in Polarized Epithelial Cells

Background

Many genome-wide association studies pointed out that SLC2A9 gene, which encodes a voltage-driven urate transporter, SLC2A9/GLUT9 (a.k.a. URATv1), as one of the most influential genes for serum urate levels. SLC2A9 is reported to encode two splice variants: SLC2A9-S (512 amino acids) and SLC2A9-L (540 amino acids), only difference being at their N-termini. We investigated isoform-specific localization of SLC2A9 in the human kidney and role of N-terminal amino acids in differential sorting in vitro.

Methodology/Principal Findings

Isoform specific antibodies against SLC2A9 were developed and human kidney sections were stained. SLC2A9-S was expressed in the apical side of the collecting duct while SLC2A9-L was expressed in the basolateral side of the proximal tubule. GFP fused SLC2A9s were expressed in MDCK cells and intracellular localization was observed. SLC2A9-S was expressed at both apical and basolateral membranes, whereas SLC2A9-L was expressed only at the basolateral membrane. Although SLC2A9-L has a putative di-leucine motif at 33th and 34th leucine, deletion of the motif or replacement of leucine did not affect its subcellular localization. When up to 16 amino acids were removed from the N-terminal of SLC2A9-S or when up to 25 amino acids were removed from the N-terminal of SLC2A9-L, there was no change in their sorting. Deletion of 20 amino acids from SLC2A9-S was not expressed in the cell. More than 30 amino acids deletion from SLC2A9-L resulted in expression at both apical and basolateral membranes as well as in the lysosome. When amino acids from 25th and 30th were changed to alanine in SLC2A9-L, expression pattern was the same as wild-type.

Conclusions/Significance

SLC2A9-L was expressed in the basolateral membrane of kidney proximal tubules in humans and this isoform is likely to responsible for urate reabsorption. N-terminal amino acids unique to each isoform played an important role in protein stability and trafficking.     

The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents. URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K(D) = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V(max) of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.     

Urate Transport Across the Apical Membrane of Renal Proximal Tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or “urate transportsome,” that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Urate transport across the apical membrane of renal proximal tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or "urate transportsome," that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.     

Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses

Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.     

Developmental expression of renal organic anion transporters in rat kidney and its effect on renal secretion of phenolsulfonphthalein

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.     

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.     

Modulation of the basolateral and apical step of transepithelial organic anion secretion in proximal tubular opossum kidney cells. Acute effects of epidermal growth factor and mitogen-activated protein kinase

The organic anion transport system in the proximal tubule of the kidney is of major importance for the excretion of a variety of endogenous and potentially toxic exogenous substances. Furthermore, the clearance of model substrates (e.g. para-aminohippurate) of this system is used for the determination of renal blood flow. We investigated regulation of organic anion secretion in a way that allowed us to examine simultaneously regulation of overall transepithelial secretion and to estimate the separate contributions of regulation of the basolateral and apical transport steps to this overall regulation. The data were verified by measurement of initial basolateral uptake rate and initial apical efflux rate. Opossum kidney cells were used as a suitable model system for proximal tubule cells, and [14C]para-aminohippurate was utilized as an organic anion. Stimulation of protein kinase C inhibited transepithelial secretion because of inhibition of both apical efflux and basolateral uptake. Inhibition of the mitogen-activated protein kinase (MAPK) kinase MEK reduced transepithelial secretion via inhibition of basolateral uptake and apical efflux. Epidermal growth factor (EGF) enhanced transepithelial secretion via stimulation of basolateral uptake but did not affect apical efflux. EGF induced stimulation of basolateral uptake was abolished by inhibition of MEK. EGF led to phosphorylation of ERK1/2, which was also abolished by inhibition of MEK. Thus, EGF stimulated basolateral uptake of organic anions via MAPKs. Transepithelial organic anion secretion can be regulated at two sites, at least: basolateral uptake and apical efflux. Both steps are under control of protein kinase C and MAPK. The pathophysiologically relevant growth factor EGF enhances transepithelial secretion via stimulation of basolateral uptake. EGF stimulates basolateral uptake via MEK and ERK1/2. Thus, renal organic anion extraction may be modulated, especially under pathophysiological conditions.     

Renal nucleoside transporters: physiological and clinical implications.

Renal handling of physiological and pharmacological nucleosides is a major determinant of their plasma levels and tissue availabilities. Additionally, the pharmacokinetics and normal tissue toxicities of nucleoside drugs are influenced by their handling in the kidney. Renal reabsorption or secretion of nucleosides is selective and dependent on integral membrane proteins, termed nucleoside transporters (NTs) present in renal epithelia. The 7 known human NTs (hNTs) exhibit varying permeant selectivities and are divided into 2 protein families: the solute carrier (SLC) 29 (SLC29A1, SLC29A2, SLC29A3, SLC29A4) and SLC28 (SLC28A1, SLC28A2, SLC28A3) proteins, otherwise known, respectively, as the human equilibrative NTs (hENTs, hENT1, hENT2, hENT3, hENT4) and human concentrative NTs (hCNTs, hCNT1, hCNT2, hCNT3). The well characterized hENTs (hENT1 and hENT2) are bidirectional facilitative diffusion transporters in plasma membranes; hENT3 and hENT4 are much less well known, although hENT3, found in lysosomal membranes, transports nucleosides and is pH dependent, whereas hENT4-PMAT is a H+-adenosine cotransporter as well as a monoamine-organic cation transporter. The 3 hCNTs are unidirectional secondary active Na+-nucleoside cotransporters. In renal epithelial cells, hCNT1, hCNT2, and hCNT3 at apical membranes, and hENT1 and hENT2 at basolateral membranes, apparently work in concert to mediate reabsorption of nucleosides from lumen to blood, driven by Na+ gradients. Secretion of some physiological nucleosides, therapeutic nucleoside analog drugs, and nucleotide metabolites of therapeutic nucleoside and nucleobase drugs likely occurs through various xenobiotic transporters in renal epithelia, including organic cation transporters, organic anion transporters, multidrug resistance related proteins, and multidrug resistance proteins. Mounting evidence suggests that hENT1 may have a presence at both apical and basolateral membranes of renal epithelia, and thus may participate in both selective secretory and reabsorptive fluxes of nucleosides. In this review, the renal handling of nucleosides is examined with respect to physiological and clinical implications for the regulation of human kidney NTs and adenosine signaling, intracellular nucleoside transport, and nephrotoxicities associated with some nucleoside drugs.     

Molecular characterization of the renal organic anion transporter 1

Organic anions of diverse chemical structures are secreted in renal proximal tubules. The first step in secretion, uptake of organic anions across the basolateral membrane of tubule cells, is mediated for the polyspecific organic anion transporter 1 (OAT1), which exchanges extracellular organic anions for intracellular α-ketoglutarate or glutarate. OAT1 orthologs cloned from various species show 12 putative transmembrane domains and possess several sites for potential post-translational modification. The gene for the human OAT1 is located on chromosome 11q13.1 and is composed of 10 exons. Alternative splicing within exon 9 gives rise to four variants, two of which (OAT1-1 and OAT1-2) are functional. Following heterologous expression in Xenopus laevis oocytes, flounder renal OAT1 transported p-aminohippurate, glutarate, several diuretics, and the nephrotoxic agent ochratoxin A. Two cationic amino acid residues, lysine 394 and arginine 478, were found to be important for interaction with glutarate. Anionic neurotransmitter metabolites and the heavy-metal chelator, 2,3-dimercaptopropane sulfonate, interacted with the rabbit renal OAT1, which is expressed in kidneys and the retina.     

Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13)

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.     

Activation of Protein Kinase C�� 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 V_max 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.Organic anion transporters (OATs)⁷ are members of the solute carrier 22A family and play a pivotal role in the renal clearance of small (<500 Dalton) anionic drugs, xenobiotics, and their metabolites. OAT substrates include a variety of drugs such as β-lactam antibiotics, non-steroidal anti-inflammatory drugs, diuretics, and chemotherapeutics (1). OATs are predominantly expressed in renal proximal tubule, with OATs 1–3 localized to the basolateral membrane and OAT4 and URAT1 on the apical membrane. OATs 1 and 3 are dicarboxylate exchangers, and are indirectly coupled to the sodium gradient maintained by Na,K-ATPase through sodium/dicarboxylate co-transport to drive the uphill basolateral step in renal organic anion secretion (2).Although the ionic gradients, electrophysiology, and underlying kinetics that drive transport by OATs 1 and 3 are well characterized, physiologically important interactions of these basolateral OATs with membrane or cytosolic proteins have yet to be identified (1). Nevertheless, there is clear evidence that other plasma membrane transporters do interact with protein partners, influencing a diverse array of functions including transport itself, cytoskeletal structure, vesicle formation, and trafficking, as well as signaling (3). Among the transporters with activity modulated by protein-protein interactions, particularly by the PDZ proteins, PDZK1 and NHERFs 1 and 2, are apical drug transporters of the SLC22A family, including OCTN1, OCTN2, OAT4, and URAT1 (4–6).In the present study, we have used a yeast two-hybrid assay to identify putative protein partners that interact directly with OAT3. The C-terminal 81 amino acids of OAT3 were used as bait to screen a human cDNA kidney library. Among the 23 positive clones (putative binding partners) was a clone encoding the C-terminal 141 amino acids of atypical protein kinase Cζ (PKCζ). Functional consequences of the putative OAT3/PKCζ interaction were investigated in rodent renal slices. The resulting data indicate that activation of PKCζ by insulin or epidermal growth factor (EGF) increased OAT3- and OAT1-mediated transport. Thus, PKCζ controls function of both major secretory organic anion transporters expressed at the basolateral face of the renal proximal tubule, positioning it to regulate the efficacy of renal drug elimination.