Browsing by Author "Bahn, Andrew"

Human trophoblasts depend on the supply of external precursors such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16 alpha-OH-DHEA-S for synthesis of estrogens. Recently, we have characterized the uptake of DHEA-S by isolated mononucleated trophoblasts and identified different transporter polypeptides involved in this process. Immunohistochemistry of 1st and 3rd trimester placenta detected organic anion transporter4 (OAT4) and organic anion transporting polypeptide 2B1 (OATP2B1, former name OATP-B) in cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast, indicating that both transporter polypeptides are involved in placental uptake of foetal derived steroid sulfates. In the present study we have characterized and compared the kinetics of DHEA-S and estrone sulfate (E1S) uptake by these transporters stably expressed in FlpIn (TM)-HEK293 cells using the Flp recombinase-mediated site-specific recombination. Uptake of EIS by OAT4- and OATP2B1-transfected cells was highly increased compared to the non-transfected cells. In contrast, DHEA-S uptake was only highly increased in OAT4 (40 times), but only weakly enhanced in OATP2B1 cells. The uptake of DHEA-S and E, S by OAT4 was partly Na+-dependent (about 50%), whereas uptake of DHEA-S by OATP2B1 was Na+-independent. Kinetic analysis of the initial uptake rates of El S by OAT4 and OATP2B1 gave very similar values for K-m (about 20 mu M) and V-max (about 600 pmol/(min x mg protein)). In contrast, the affinity of DHEA-S towards OATP2B1 was about 10 times lower (Km > 200 mu M) then for OAT4 (K-m = 29 mu M). Our results suggest different physiological roles of the two transporter polypeptides in placental uptake of foetal derived steroid sulfates. OATP2B1 seems not to be involved in de novo synthesis of placental estrogens but may contribute to the clearance of estrogen sulfates from foetal circulation. (C) 2008 Elsevier Ltd. All rights reserved.

Sex hormones influence the development of female (F) and male (M) specific traits and primarily affect the structure and function of gender-specific organs. Recent studies also indicated their important roles in regulating structure and/or function of nearly every tissue and organ in the mammalian body, including the kidneys, causing gender differences in a variety of characteristics. Clinical observations in humans and studies in experimental animals in vivo and in models in vitro have shown that renal structure and functions under various physiological, pharmacological, and toxicological conditions are different in M and F, and that these differences may be related to the sex-hormone-regulated expression and action of transporters in the apical and basolateral membrane of nephron epithelial cells. In this review we have collected published data on gender differences in renal functions, transporters and other related parameters, and present our own microarray data on messenger RNA expression for various transporters in the kidney cortex of M and F rats. With these data we would like to emphasize the importance of sex hormones in regulation of a variety of renal transport functions and to initiate further studies of gender-related differences in kidney structure and functions, which would enable us to better understand occurrence and development of various renal diseases, pharmacotherapy, and drug-induced nephrotoxicity in humans and animals.

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 mu M. 6-CF as well as [H-3]estrone sulfate ([H-3]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 [H-3]ES uptake, indicating an asymmetric interaction of hOAT4 with these substrates. In chloride-free medium, HEK293-hOAT4-mediated [H-3]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 [H-3]ES uptake, which was reduced by nigericin, suggesting that hOAT4 also can operate as an OA/OH- exchanger. hOAT4 facilitates substantial uptake of [C-14]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.

Aristolochic acid (AA), present in Aristolochia species, is the major causative agent in the development of severe renal failure and urothelial cancers in patients with AA nephropathy. It may also be a cause of Balkan endemic nephropathy. Epithelial cells of the proximal tubule are the primary cellular target of AA. To study whether organic anion transporters (OATs) expressed in proximal tubule cells are involved in uptake of AA, we used human epithelial kidney (HEK293) cells stably expressing human (h) OAT1, OAT3 or OAT4. AA potently inhibited the uptake of characteristic substrates, p-aminohippurate for hOAT1 and estrone sulfate for hOAT3 and hOAT4. Aristolochic acid I (AAI), the more cytotoxic and genotoxic AA congener, exhibited high affinity for hOAT1 (K(i) = 0.6 mu M) as well as hOAT3 (K(i) = 0.5 mu M), and lower affinity for hOAT4 (K(i) = 20.6 mu M). Subsequently, AAI-DNA adduct formation (investigated by (32)P-postlabelling) was used as a measure of AAI uptake. Significantly higher levels of adducts occurred in hOAT-expressing cells than in control cells: this effect was abolished in the presence of the OAT inhibitor probenecid. In Xenopus laevis oocytes hOAT-mediated efflux of p-aminolhippurate was trans-stimulated by extracellular AA, providing further molecular evidence for AA translocation by hOATs. Our study indicates that OATs can mediate the uptake of AA into proximal tubule cells and thereby participate in kidney cell damage by this toxin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Recent investigations involving intact rabbit renal proximal tubules indicated that organic anion transporter 3 (OAT3) may be involved in the transport of 2,3-dimercapto-1-propanesulfonic acid (DMPS). Therefore, we evaluated the interaction of OAT3 with DMPS to determine the effect of OAT3 on basolateral DMPS uptake. We used stably transfected HEK293 cells expressing human and rabbit orthologs of the exchanger OAT1 and OAT3. Using 6-carboxyfluorescein (6-CF) as a substrate, the IC(50) determinations for reduced DMPS (DMPSH) revealed a stronger interaction with OAT1 than with OAT3 (rbOAT1, 123.3 +/- 13.7: hOAT1, 85.1 +/- 8.8; rbOAT3, 171.7 +/- 22.3: and hOAT3, 172.2 +/- 36.4 mu mol/L). However, inhibition of 6-CF uptake by the oxidized form of DMPS (DMPSS), the main form of DMPS in the blood, showed a greater affinity for OAT3 (rbOAT1, 237.4 +/- 23; hOAT1, 104.6 +/- 13.1: rbOAT3, 52.4 +/- 7.6: and hOAT3, 31.6 +/- 6.6 mu mol/L). To determine whether DMPSH and DMPSS are substrates for OAT3, we performed efflux studies with [(14)C]glutarate and inwardly directed gradients of glutarate. The inhibitors trans-stimulated the efflux of [(14)C]glutarate, suggesting that OAT3 may be able to transport both forms of DMPS. On the basis of the substantial interaction of OAT3 with DMPSS, we conclude that OAT3 represents the dominant basolateral player in renal detoxification processes resulting from use of DMPS.

The renal reabsorption and/or excretion of various organic anions is mediated by specific organic anion transporters (OATs). OAT2 (Slc22a7) has been identified in rat kidney, where its mRNA expression exhibits gender differences [females (F) > males (M)]. The exact localization of OAT2 protein in the mammalian kidney has not been reported. Here we studied the expression of OAT2 mRNA by RT-PCR and its protein by Western blotting (WB) and immunocytochemistry (IC) in kidneys of adult intact and gonadectomized M and F, sex hormone-treated castrated M, and prepubertal M and F rats, and the protein in adult M and F mice. In adult rats, the expression of OAT2 mRNA was predominant in the outer stripe (OS) tissue, exhibiting 1) gender dependency (F > M), 2) upregulation by castration and downregulation by ovariectomy, and 3) strong downregulation by testosterone and weak upregulation by estradiol and progesterone treatment. A polyclonal antibody against rat OAT2 on WB of isolated renal membranes labeled a similar to 66-kDa protein band that was stronger in F. By IC, the antibody exclusively stained brush border (BB) of the proximal tubule S3 segment (S3) in the OS and medullary rays (F > M). In variously treated rats, the pattern of 66-kDa band density in the OS membranes and the staining intensity of BB in S3 matched the mRNA expression. The expression of OAT2 protein in prepubertal rats was low and gender independent. In mice, the expression pattern largely resembled that in rats. Therefore, OAT2 in rat (and mouse) kidney is localized to the BB of S3, exhibiting gender differences (F > M) that appear in puberty and are caused by strong androgen inhibition and weak estrogen and progesterone stimulation.

The high renal clearance of torasemide, the most potent loop diuretic, suggests active tubular secretion in the proximal tubule. Previous studies implicated the organic anion transporters (OAT) in this process; human OAT1 (hOAT1) and hOAT3 are found on the basolateral surface of proximal tubule cells, and hOAT4 is found on the luminal surface. This study sought to determine the mechanism underlying renal elimination of torasemide and to elucidate the drug's effect on renal urate secretion, because hyperuricemia is a concerning adverse effect. Torasemide and its metabolites were transported into stably transfected HEK293 cells by hOAT1, hOAT3, and hOAT4 and out of the cells by hOAT3 and hOAT4. These data suggest that basolateral hOAT3 and luminal hOAT4 are likely responsible for the translocation of torasemide across the proximal tubule cell. Regarding urate handling, torasemide and its metabolites did not interact with human URAT1, but competitive inhibition of the basolateral OAT for urate may reduce tubular secretion. Furthermore, because hOAT4 can reabsorb urate from the urinary lumen, increased urate reabsorption may occur as exchange for the secretion of torasemide and its metabolites. In support of this hypothesis, fractional excretion of urate was reduced in 95 healthy volunteers after torasemide administration. In summary, this study determined the affinity of OAT for torasemide and its metabolites and proposed a mechanism underlying torasemide-induced hyperuricemia that does not involve the human URAT1-mediated transport affected by other loop diuretics.

The procarcinogen 1-methylpyrene is activated by hepatic enzymes via 1-hydroxymethylpyrene to 1-sulfooxymethylpyrene (1-SMP), a highly reactive and mutagenic metabolite. Previously, high levels of 1-SMP DNA adducts were observed in rat kidneys after intraperitorteal administration of 1-hydroxymethylpyrene or 1-SMP. This study examined whether organic anion transporters (OAT) that are expressed at the basolateral membrane of proximal tubule cells are involved in uptake of SMP. Human epithelial kidney (HEK293) cells that stably express human OAT1 (hOAT1) and hOAT3 were used. Stable isomers of 1-SMP, (2-SMP and 4-SMP) competitively inhibited the uptake of characteristic substrates p-aminohippurate for hOAT1 and estrone sulfate for hOAT3. Both inhibitors exhibited high affinity for hOAT1 (K-i = 4.4 mu M for 2-SMP; Ki = 5.1 mu M for 4-SMP) as well as hOAT3 (K-i = 1.9 mu M for 2-SMP; K-i = 2.1 mu M for 4-SMP). The uptake rate of 4-SMP (at a concentration of 10 mu M) by hOAT1- and hOAT3-expressing cells was 3.0 and 1.6 times higher, respectively, than in control cells. Uptake of the reactive isomer 1-SMP was investigated using as the end point the level of DNA adducts that were formed in the cells. After exposure to 1-SMP (10 mu M), the DNA adduct level was 4.6 and 3.0 times higher in hOAT1- and hOAT3-expressing cells, respectively, than in control cells. The enhanced DNA adduct formation in hOAT-expressing cells was abolished in the presence of the OAT inhibitor probenecid. This study indicates that OAT can mediate the basolateral uptake of reactive sulfuric acid esters into proximal tubule cells and thereby participate in kidney cell damage by these compounds.

Background. Hereditary renal hypouricemia may be complicated by nephrolithiasis or exercise-induced acute renal failure. Most patients described so far are of Japanese origin and carry the truncating mutation W258X in the uric acid transporter URAT1 encoded by SLC22A12. Recently, we described severe renal hypouricemia in Israeli patients with uric acid transporter GLUT9 (SLC2A9) loss-of-function mutations. Renal hypouricemia in Iraqi Jews has been previously reported, but its molecular basis has not been ascertained. Methods. Three Jewish Israeli families of Iraqi origin with hereditary hypouricemia and hyperuricosuria were clinically characterized. DNA was extracted and the URAT1 gene was sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of URAT1 mutants found. Results. A missense URAT1 mutation, R406C, was detected in all three families. Two affected siblings were found to carry in addition a homozygous missense URAT1 mutation, G444R. Both mutations dramatically impaired urate uptake into X. laevis oocytes. Moreover, we demonstrate for the first time that URAT1 facilitates urate efflux, which was abolished in the mutants, indicating also a secretion defect. Homozygous patients had serum uric acid concentrations of 0.5-0.8 mg% and a fractional excretion of uric acid of 50-85%. Most individuals studied were asymptomatic, two had nephrolithiasis and none developed exercise-induced acute renal failure. Conclusions. The URAT1 R406C mutation detected in all three families is likely to be the founder mutation in Iraqi Jews. Our findings contribute to a better definition of the different types of hereditary renal hypouricemia and suggest that the phenotype of this disorder depends mainly on the degree of inhibition of uric acid transport.