Birgitta C. Burckhardt

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 [3H]nicotinate, [3H]p-aminohippurate, and [14C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [3H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (Km) of 22 and 44 μm, 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 [3H]nicotinate transport by hOAT10 with an IC50 value of 759 μm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [14C]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 [14C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.

In Vitro and In Vivo Evidence of the Importance of Organic Anion Transporters (OATs) in Drug Therapy

Organic anion transporters 1-10 (OAT1-10) and the urate transporter 1 (URAT1) belong to the SLC22A gene family and accept a huge variety of chemically unrelated endogenous and exogenous organic anions including many frequently described drugs. OAT1 and OAT3 are located in the basolateral membrane of renal proximal tubule cells and are responsible for drug uptake from the blood into the cells. OAT4 in the apical membrane of human proximal tubule cells is related to drug exit into the lumen and to uptake of estrone sulfate and urate from the lumen into the cell. URAT1 is the major urate-absorbing transporter in the apical membrane and is a target for uricosuric drugs. OAT10, also located in the luminal membrane, transports nicotinate with high affinity and interacts with drugs. Major extrarenal locations of OATs include the blood-brain barrier for OAT3, the placenta for OAT4, the nasal epithelium for OAT6, and the liver for OAT2 and OAT7. For all transporters we provide information on cloning, tissue distribution, factors influencing OAT abundance, interaction with endogenous compounds and different drug classes, drug/drug interactions and, if known, single nucleotide polymorphisms.

The Renal Na+-Dependent Dicarboxylate Transporter, NaDC-3, Translocates Dimethyl- and Disulfhydryl-Compounds and Contributes to Renal Heavy Metal Detoxification

The active transport of Krebs cycle intermediates, such as succinate, alpha-ketoglutarate, and citrate, is mediated by sodium-coupled transporters found in the luminal (NaDC-1) and basolateral plasma membranes (NaDC-3) of proximal tubule cells. This study used the two-electrode voltage clamp technique to examine steady-state currents associated with the influx of three sodium ions and one divalent dicarboxylate into oocytes expressing the sodium-dicarboxylate transporter from winter flounder kidney, fNaDC-3. The substrate concentration, where half-maximal current was observed (K(0.5)), was 30 micro M for succinate. Besides 2,2-dimethylsuccinate, fNaDC-3 also accepted 2,3-dimethylsuccinate and the oral lead-chelating agent, meso-2,3-dimercaptosuccinate (DMSA or Succimer). Whereas the K(0.5) for succinate and 2,2-dimethylsuccinate was independent of membrane voltage within -90 and -10 mV, K(0.5) for 2,3-dimethylsuccinate and 2,3-dimercaptosuccinate increased with decreasing voltage, indicating a critical role of the position of the methyl- or sulfhydryl-group in voltage-sensitive affinity. In addition to meso-2,3-dimercaptosuccinate, fNaDC-3 translocated dimercaptopropane-1-sulfonate (DMPS or Dimaval), an oral chelator for the treatment of mercury intoxication. The chelates formed by HgCl(2) and DMSA or DMPS and by Pb(NO(3))(2) and DMSA, however, were not translocated by fNaDC-3. The data suggest that NaDC-3 is an essential component in the delivery of uncomplexed antidotes for renal heavy metal detoxification.

NH4+ conductance in Xenopus laevis oocytes

Abstract Current-clamp and voltage-clamp techniques were used to study the effects of NH4+ on the cell membrane conductance in Xenopus laevis oocytes. Superfusing the oocytes with NH4Cl resulted in a depolarization of the oocyte’s cell membrane potential and, at a clamp potential of –70 mV, in an inward current. The magnitude of the inward current was proportional to the NH4Cl concentration in the extracellular solution and on membrane potential. The reversal potential, Erev , was –35.5 ± 11.6 mV under control conditions and –3.1 ± 11.0 mV (n = 19) in the presence of NH4Cl (10 mmol/l). Superfusion of the oocytes with nominally Ca2+-free solution affected the NH4Cl-evoked response only marginally. Replacement of extracellular Na+ by N-methyl-D-glucamine+ markedly reduced, but did not eliminate, the NH4Cl-sensitive current and shifted the reversal potential to more negative potentials. The NH4Cl-induced current was substantially inhibited by 0.1 mmol/l flufenamate, and was less affected by blockers of the endogenous K+ conductance, Ba2+ and isosorbiddinitrate (ISDN). The results are compatible with the activation of a conductance by NH4Cl for Na+ and NH4+. The mechanism by which NH4Cl activates the conductance remains unknown.

Expression of the human sodium/proton exchanger NHE-1 in Xenopus laevis oocytes enhances sodium/proton exchange activity and establishes sodium/lithium countertransport

We investigated whether the human sodium/proton (Na+/H+) exchanger isoform 1 (NHE-1) can mediate sodium/lithium (Na+/Li+) coutertransport. Using the Xenopus laevis oocyte expression system we determined amiloride-sensitive Li+ uptake, a measure of Na+/H+ exchange, in oocytes injected with water or NHE-1 cRNA. Amiloride-sensitive Li+ uptake was three-to tenfold enhanced over control in NHE-1 cRNA-injected cells and was selectively inhibited by 0.01 μM HOE 694 [i.e. (3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate]. The endogenously present Na+/H+ exchanger was insensitive to HOE 694. After acidification of oocytes from pH 7.7 to 6.8, amiloride-sensitive Li+ uptake was four-to tenfold higher in NHE-1 cRNA-injected cells than in controls. Li+ efflux from control oocytes was independent of extracellular Na+, indicating that these cells expressed no measurable Na+/Li+ countertransport activity. In NHE-1 cRNA-injected oocytes, Li+ efflux was distinctly enhanced by extracellular Na+ ions. This Na+-dependent Li+ efflux was inhibited by ethylisopropylamiloride, phloretin and by cytosolic acidification. The data show that expression of the NHE-1 in X. laevis oocytes induces the expression of Na+/Li+ countertransport. The data confirm that Na+/H+ exchange and Na+/Li+ countertransport are mediated by the same transport system.

Expression Cloning and Characterization of a Novel Sodium-Dicarboxylate Cotransporter from Winter Flounder Kidney

A cDNA coding for a Na+-dicarboxylate cotransporter, fNaDC-3, from winter flounder (Pseudopleuronectes americanus) kidney was isolated by functional expression in Xenopus laevisoocytes. The fNaDC-3 cDNA is 2384 nucleotides long and encodes a protein of 601 amino acids with a calculated molecular mass of 66.4 kDa. Secondary structure analysis predicts at least eight membrane-spanning domains. Transport of succinate by fNaDC-3 was sodium-dependent, could be inhibited by lithium, and evoked an inward current. The apparent affinity constant (K m ) of fNaDC-3 for succinate of 30 μm resembles that of Na+-dicarboxylate transport in the basolateral membrane of mammalian renal proximal tubules. The substrates specific for the basolateral transporter, 2,3-dimethylsuccinate and cis-aconitate, not only inhibited succinate uptake but also evoked inward currents, proving that they are transported by fNaDC-3. Succinate transport via fNaDC-3 decreased by lowering pH, as did citrate transport, although much more moderately. These characteristics suggest that fNaDC-3 is a new type of Na+-dicarboxylate transporter that most likely corresponds to the Na+-dicarboxylate cotransporter in the basolateral membrane of mammalian renal proximal tubules.

Effect of primary, secondary and tertiary amines on membrane potential and intracellular pH in Xenopus laevis oocytes.

The effects of primary, secondary and tertiary methyl- and ethylamines as well as of quaternary ammonium compounds on membrane potential, Vm, and intracellular pH (pHi) of oocytes from Xenopus laevis were studied using electrophysiological methods. The quaternary ammonium compounds, tetramethyl- (TMA) and tetraethyl- (TEA) ammonium chloride and choline chloride (each 10 mmol/l), affected Vm only slightly. In contrast, primary, secondary and tertiary amines strongly depolarized Vm. Depolarization was inversely proportional to the pKa of the amines. Trimethylamine (pKa 9.8) depolarized Vm by 61.7±21.8 mV (n=13) and exerted its half-maximal effect at less than 2 mmol/l. In paired experiments (n=6), trimethylamine (10 mmol/l) reduced Vm only by 5.1±1.3 mV at a bath pH of 6.0, but by 73.2±20.0 mV at pH 7.5, suggesting that the deprotonated, uncharged form of the amines was responsible for the depolarization. pHi measurements using the Fluka pH-sensitive cocktail 95 293 revealed a short initial alkalinization and a subsequent acidification in the presence of trimethylamine (10 mmol/l). The intracellular acidification proceeded much more slowly than the depolarization. As shown by measurements using a two-electrode voltage-clamp device, the depolarization was associated with an inward current. This trimethylamine-sensitive current, ΔIm, decreased from-128±82 nA (n=4) at a clamp potential Vc=-70 mV to-3±33 nA at Vc=0 mV. Neither ΔVm nor ΔIm were markedly inhibited by GdCl3, BaCl2, or amiloride (each 1 mmol/l). Only 1 mmol/l diphenylamine-2-carboxylate (DPC) diminished both responses. The data suggest that the amines modify anion or cation conductances of the oocytes by as yet unknown mechanisms.

Organic anion transporters OAT1 and OAT4 mediate the high affinity transport of glutarate derivatives accumulating in patients with glutaric acidurias

Glutaric acidurias are rare inherited neurodegenerative disorders accompanied by accumulation of the metabolites glutarate (GA) and 3-hydroxyglutarate (3OHGA), glutaconate, L-, or D-2-hydroxyglutarate (L-2OHGA, D-2OHGA) in all body fluids. Oocytes expressing the human (h) sodium-dicarboxylate cotransporter (NaDC3) showed sodium-dependent inward currents mediated by GA, 3OHGA, L-, and D-2OHGA. The organic anion transporters (OATs) were examined as additional transporters for GA derivatives. The uptake of [3H]p-aminohippurate in hOAT1-transfected human embryonic kidney (HEK293) cells was inhibited by GA, 3OHGA, D-, or L-2OHGA in a concentration-dependent manner. None of these compounds affected the hOAT3-mediated uptake of [3H]estrone sulfate (ES). In hOAT4-expressing cells and oocytes, ES uptake was strongly increased by intracellular GA derivatives. The data provide a model for the concerted action of OAT1 and NaDC3 mediating the basolateral uptake, and OAT4 mediating apical secretion of GA derivatives from proximal tubule cells and therefore contribute to the renal clearance of these compounds.

Ability of sat-1 to transport sulfate, bicarbonate, or oxalate under physiological conditions

Tubular reabsorption of sulfate is achieved by the sodium-dependent sulfate transporter, NaSi-1, located at the apical membrane, and the sulfate-anion exchanger, sat-1, located at the basolateral membrane. To delineate the physiological role of rat sat-1, [(35)S]sulfate and [(14)C]oxalate uptake into sat-1-expressing oocytes was determined under various experimental conditions. Influx of [(35)S]sulfate was inhibited by bicarbonate, thiosulfate, sulfite, and oxalate, but not by sulfamate and sulfide, in a competitive manner with K(i) values of 2.7 +/- 1.3 mM, 101.7 +/- 9.7 microM, 53.8 +/- 10.9 microM, and 63.5 +/- 38.7 microM, respectively. Vice versa, [(14)C]oxalate uptake was inhibited by sulfate with a K(i) of 85.9 +/- 9.5 microM. The competitive type of inhibition indicates that these compounds are most likely substrates of sat-1. Physiological plasma bicarbonate concentrations (25 mM) reduced sulfate and oxalate uptake by more than 75%. Simultaneous application of sulfate, bicarbonate, and oxalate abolished sulfate as well as oxalate uptake. These data and electrophysiological studies using a two-electrode voltage-clamp device provide evidence that sat-1 preferentially works as an electroneutral sulfate-bicarbonate or oxalate-bicarbonate exchanger. In kidney proximal tubule cells, sat-1 likely completes sulfate reabsorption from the ultrafiltrate across the basolateral membrane in exchange for bicarbonate. In hepatocytes, oxalate extrusion is most probably mediated either by an exchange for sulfate or bicarbonate.

Selective Stabilization of HIF-1α in Renal Tubular Cells by 2-Oxoglutarate Analogues

The role of proximal versus distal tubular injury in the pathogenesis of acute kidney injury (AKI) is debatable. Inhibition of prolyl hydroxylases that regulate the degradation of hypoxia-inducible transcription factors (HIFs) is a promising therapeutic approach to optimize energy preservation under hypoxia and has successfully been applied to protect kidney structure and function in AKI models. Presently used prolyl hydroxylase inhibitors are lipophilic 2-oxoglutarate analogues (2OGAs) that are widely taken up in cells of most organs. Given the selective expression of organic anion transporters (OATs) in renal proximal tubular cells, we hypothesized that hydrophilic 2OGAs can specifically target proximal tubular cells. We found that cellular hydrophilic 2OGAs uptake depended on OATs and largely confined to the kidney, where it resulted in activation of HIF target genes only in proximal tubular cells. When applied in ischemia-reperfusion experiments, systemically active 2OGA preserved kidney structure and function, but OAT1-transported 2OGA was not protective, suggesting that HIF stabilization in distal tubular rather than proximal tubular cells and/or nontubular cells mediates protective effects. This study provides proof of concept for selective drug targeting of proximal tubular cells on the basis of specific transporters, gives insights into the role of different nephron segments in AKI pathophysiology, and may offer options for long-term HIF stabilization in proximal tubules without confounding effects of erythropoietin induction in peritubular cells and unwarranted extrarenal effects.