Yohannes Hagos

Human Renal Organic Anion Transporter 4 Operates as an Asymmetric Urate Transporter

Human organic anion transporter 4 (hOAT4) is located at the apical membrane of proximal tubule cells and involved in renal secretion and reabsorption of endogenous substances as well as many drugs and xenobiotics. This study reevaluated the physiologic role, transport mode, and driving forces of hOAT4. 6-Carboxyfluorescein (6-CF) uptake into HEK293 cells that stably expressed hOAT4 was saturable, resulting in a K(m) of 108 muM. 6-CF as well as [(3)H]estrone sulfate ([(3)H]ES) accumulation by HEK293-hOAT4 cells were abolished by ES, dehydroepiandrosterone sulfate, sulfinpyrazone, benzbromarone, and probenecid, whereas several OA, including p-aminohippurate (PAH), lactate, pyrazinoate, nicotinate, glutarate, and the diuretic hydrochlorothiazide (HCTZ) exhibited a slight or a NS inhibitory effect. PAH and glutarate are not taken up by HEK293-hOAT4 cells, but they trans-stimulated 6-CF and [(3)H]ES uptake, indicating an asymmetric interaction of hOAT4 with these substrates. In chloride-free medium, HEK293-hOAT4-mediated [(3)H]PAH efflux was almost abolished, whereas addition of ES restored it comparable to Ringer solution, consistent with a physiologic ES/PAH or PAH/Cl(-) exchange mode of hOAT4. Moreover, an acidification of the uptake medium increased 6-CF as well as [(3)H]ES uptake, which was reduced by nigericin, suggesting that hOAT4 also can operate as an OA/OH(-) exchanger. hOAT4 facilitates substantial uptake of [(14)C]urate, which was elevated 2.6-fold by intracellular HCTZ. Thus, hOAT4 is the long-postulated, low-affinity apical urate anion exchanger that facilitates HCTZ-associated hyperuricemia.

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.

Assessment of the Role of Renal Organic Anion Transporters in Drug-Induced Nephrotoxicity

In the present review we have attempted to assess the involvement of the organic anion transporters OAT1, OAT2, OAT3, and OAT4, belonging to the SLC22 family of polyspecific carriers, in drug-induced renal damage in humans. We have focused on drugs with widely recognized nephrotoxic potential, which have previously been reported to interact with OAT family members, and whose underlying pathogenic mechanism suggests the participation of tubular transport. Thus, only compounds generally believed to cause kidney injury either by means of direct tubular toxicity or crystal nephropathy have been considered. For each drug, or class of agents, the evidence for actual transport mediated by individual OATs under in vivo conditions is discussed. We have then examined their role in the context of other carriers present in the renal proximal tubule sharing certain substrates with OATs, as these are critical determinants of the overall contribution of OAT-dependent transport to intracellular accumulation and transepithelial drug secretion, and thus the impact it may have in drug-induced nephrotoxicity.

Human Organic Cation Transporter 1 Is Expressed in Lymphoma Cells and Increases Susceptibility to Irinotecan and Paclitaxel

Antineoplastic agents directed at nuclear and cytoplasmic targets in tumor cells represent the current mainstay of treatment for patients with disseminated malignant diseases. Cellular uptake of antineoplastics is a prerequisite for their efficacy. Five of six lymphoma cell lines as well as primary samples from chronic lymphocytic leukemia patients demonstrated significant expression of SLC22A1 mRNA coding for organic cation transporter 1 (OCT1). Functionally, the antineoplastic agents irinotecan, mitoxantrone, and paclitaxel inhibited the uptake of the organic cation [3H]1-methyl-4-pyridinium iodide into OCT1-transfected Chinese hamster ovary model cells, with Ki values of 1.7, 85, and 50 μM, respectively. Correspondingly, OCT1-positive cell lines and transfectants exhibited significantly higher susceptibilities to the cytotoxic effects of irinotecan and paclitaxel compared with those of OCT1-negative controls. We hypothesize that OCT1 can contribute to the susceptibility of cancer cells to selected antineoplastic drugs. In the future, an expression analysis of the transporters and the application of transporter-specific antineoplastic agents could help to tailor cancer therapy.

Expression of Human Organic Cation Transporter 3 in Kidney Carcinoma Cell Lines Increases Chemosensitivity to Melphalan, Irinotecan, and Vincristine

Renal cell carcinoma (RCC) is usually chemoresistant. This chemoresistance could be overcome if specific cytostatics are applied for which the RCC expresses an uptake transporter. In the present study, we investigated the expression of solute carrier (SLC) transporters in different RCC lines and their ability to interact with chemotherapeutics. We tested five RCC lines for the expression of different SLCs by reverse transcription-PCR and TaqMan real-time PCR. In two of five RCC lines, A498 and 7860, we observed a highly significant expression of SLC22A3 (hOCT3). Uptake of the organic cation [(3)H]MPP (4-methyl-pyridinium iodide) into these cells and also into hOCT3 stably transfected Chinese hamster ovary (CHO) cells was inhibited by irinotecan, vincristine, and melphalan. The K(i) values [determined from Dixon plots] for irinotecan, vincristine, and melphalan were 1.72 +/- 0.45 micromol/L, 17 +/- 4.81 micromol/L, and 366 +/- 51 micromol/L, respectively. Cytotoxic activities of the selected drugs were tested by [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on CHO-hOCT3, A498 (high expression of hOCT3), and ACHN cell lines (low expression of hOCT3). The growth of CHO-hOCT3 was inhibited by 20% more with irinotecan and by 50% more with vincristine compared with nontransfected CHO cells. Melphalan produced 20% to 30% more inhibition in hOCT3-expressing cells compared with nonexpressing control cells. Similar results were obtained for A498 and ACHN cells. Thus, our data support the hypothesis that the sensitivity of tumor cells to chemotherapeutic treatment depends on the expression of transporter proteins mediating specific drug accumulation into target cells.

The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes

Sulforhodamine 101 (SR101) is widely used for astrocyte identification, though the labeling mechanism remains unknown and the efficacy of labeling in different brain regions is heterogeneous. By combining region-specific isolation of astrocytes followed by transcriptome analysis, two-photon excitation microscopy, and mouse genetics, we identified the thyroid hormone transporter OATP1C1 as the SR101-uptake transporter in hippocampus and cortex.

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines.

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r(2)=0.47, P=0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r(2)=0.56, P=0.03 and r(2)=0.62, P=0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA. LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.

Cloning of the pig renal organic anion transporter 1 (pOAT1).

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.

Active Sulforhodamine 101 Uptake into Hippocampal Astrocytes

Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrolateral medulla. Although carbenoxolone is able to decrease the SR101-labeling of astrocytes in the hippocampus, it is unlikely that SR101 is taken up via gap-junction hemichannels because mefloquine, a blocker for pannexin and connexin hemichannels, was unable to prevent SR101-labeling of hippocampal astrocytes. However, SR101-labeling of the hippocampal astrocytes was significantly reduced by substrates of organic anion transport polypeptides, including estron-3-sulfate and dehydroepiandrosterone sulfate, suggesting that SR101 is actively transported into hippocampal astrocytes.

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.