Differential gene expression of organic anion transporters in male and female rats.

Sex-related differential gene expression of organic anion transporters (rOAT1, rOAT2, and rOAT3) in rat brain, liver, and kidney was investigated. There were no sex differences in the expression of rOAT1 mRNA. rOAT2 mRNA was abundant in the liver and weakly expressed in the kidney of male rats; however, the OAT2 gene was strongly expressed in both organs of females. The abundance of rOAT2 mRNA markedly increased in castrated male rat kidney; however, treatment of castrated male rats with testosterone led to a decrease of rOAT2 mRNA. Expression of rOAT3 mRNA in intact female rats was found in the kidney and brain, whereas in males rOAT3 mRNA was also found in the liver. rOAT3 mRNA markedly decreased in the liver of castrated male rats but increased in testosterone-treated castrated male rats. Moreover, rOAT3 mRNA increased in the hypophysectomized female rat liver, indicating that rOAT3 is an inducible isoform. The present findings suggest that sex steroids play an important role in the expression and maintenance of OAT2/3 isoforms in the rat liver and kidney. Our results provide information on the differential gene expression of OAT isoforms with sex hormone dependency.     

Renal excretion of perfluorooctanoic acid in male rats: Inhibitory effect of testosterone

There is a marked sex difference in the whole-body elimination of perfluorooctanoic acid (PFOA) in rats, with females excreting the perfluorinated acid much more rapidly (half life [t_1/2] < 1 day) than males (t_1/2=15 days). Our objective was to determine if androgens or estrogens are involved in causing this sex difference in PFOA elimination. Castration of males greatly increased the elimination of [1-¹⁴C]PFOA (9.4 μmiol/kg, i.p.) in urine, demonstrating that a factor produced by the testis was responsible for the slow elimination of PFOA in male rats. Castration plus 17β-estradiol had no further effect on PFOA elimination whereas castration plus testosterone replacement at the physiologic level reduced PFOA elimination to the same level as rats with intact testes. Thus, in male rats, testosterone exerts an inhibitory effect on renal excretion of PFOA. In female rats, neither ovariectomy nor ovariectomy plus testosterone affected the PFOA urinary elimination, demonstrating that the inhibitory effect of testosterone on PFOA renal excretion is a male-specific response. Probenecid decreased the high rate of PFOA renal excretion in castrated males but had no effect on male rats with intact testes. We conclude that testosterone is a key determinant of the sex difference in PFOA elimination in rats.     

Characterization of inducible nature of MRP3 in rat liver

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na(+)-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. alpha-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or alpha-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.     

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.     

Hormonal regulation of organic cation transporter OCT2 expression in rat kidney

Rat (r) OCT2 was identified as the second member of the organic cation transporter (OCT) family, and is predominantly expressed in the kidney. We reported previously that rOCT2 was responsible for the gender differences in renal basolateral membrane organic cation transport activity. As renal rOCT2 expression in males is much higher than that in females, we hypothesized that rOCT2 expression may be under the control of sex hormones. Treatment of male and female rats with testosterone significantly increased the expression levels of rOCT2 mRNA and protein in the kidney, whereas estradiol treatment moderately decreased the expression levels of rOCT2. There was no regulation of renal rOCT1 mRNA expression by testosterone or estradiol. Treatment of male and female rats with testosterone significantly stimulated the tetraethylammonium (TEA) accumulation by renal slices, whereas estradiol treatment caused a decrease in the TEA accumulation by slices from male but not female rats. The present findings suggested that testosterone up-regulates renal rOCT2 expression and estradiol moderately down-regulates rOCT2.     

Effects of estradiol and testosterone on the activity of pyruvate kinase of the cardiac and skeletal muscles of rats as a function of age and sex

The specific activities of pyruvate kinase of cardiac and skeletal (gastrocnemius) muscles of adult rats of both sexes are lower than those of immature rats. The activity does not change after adulthood in the cardiac muscle, but decreases in the gastrocnemius. The activity of pyruvate kinase of the heart of immature and adult rats of both sexes decreases after castration, but is unaffected in old rats. Castration has no effect on the activity of pyrovate kinase of the gastrocnemius muscle of rats of both sexes at any age. In invo administration of estradiol (50 μg/100 g body weight) increases the activity of pyruvate kinase of the heart of castrated male and female rats of the three ages. For the skeletal muscle, the activity increases in castrated adult female and old male rats only. A higher dose (100 μg) of estradiol has variable effects on pyruvate kinase of the heart of male and female castrated rats of different ages. This dose increase pyruvate kinase significantly in the skeletal muscle of old castrated male and female rats. However, it decreases it in the skeletal muscle of adult castrated male rats. Testosterone (100 μm) increases the activity of pyruvate kinase of the heart of castrated male rats. This increase is lower in old age. It has no effect in the heart of castrated female rats of any age. Testosterone (50 μg) increases pyruvate kinase activity of the skeletal muscle of young ovariectomized rats only. A higher dose (100 μg) causes a significant increase in pyruvate kinase of the skeletal muscle of castrated adult and old male, and young and adult female rats, respectively. These data show that sex steroid hormones induce pyruvate kinase of striated muscles, and that the age- and sex-dependent variations may be due to changes in the levels of receptor proteins.     

Functional analysis of rat renal organic anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane.

Renal organic anion transporter OAT-K1 was stably transfected in MDCK cells and examined for its transport characteristics and membrane localization. OAT-K1 mediated both uptake and efflux of methotrexate in the apical membranes. Immunoblotting showed that the apparent molecular mass of the expressed OAT-K1 was 50 kDa, which was comparable to that found in the rat renal brush-border membranes. The OAT-K1-mediated methotrexate transport was significantly inhibited in the presence of several organic anions such as folate and sulfobromophthalein. These findings suggest that OAT-K1 mediates bidirectional methotrexate transport across the apical membranes, and may be involved in the renal handling of methotrexate.     

The effect of sex hormones on the acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in rat liver

The intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity (PEPCK) was investigated in microdissected samples of livers from normal, castrated, castrated and estradiol- or testosterone-treated, and uncastrated and testosterone- or estradiol-treated male and female rats. The total PEPCK activity showed a marked sex dependency, with 1.8 times higher activity in males. The intra-acinar distribution profiles were also sex-dependent. The periportal-to-perivenous gradient was steeper in males. Castration resulted in an approximation of PEPCK activity and its acinar distribution pattern between the sexes due to a reduction in males and an increase in females. Estrogen treatment of castrated males had no further effect on PEPCK activity and its acinar gradient, whereas in ovariectomized animals the activity was reduced to levels near normal. Testosterone treatment of castrated male or female animals led to a marked increase in enzyme activity with a concomitant steepening of the acinar gradient. Administration of estradiol to normal male rats also led to a reduction in activity, together with a change in the acinar activity gradient. Testosterone treatment of normal females resulted in an induction of PEPCK activity which was most prominent in the periportal zone. The most drastic changes were observed in the perivenous zones. In all experiments a periportal-to-perivenous activity gradient persisted thus marking the periportal zone as the area with highest gluconeogenic capacity.     

Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats

Elimination in urine and feces was compared between four perfluorinated fatty acids (PFCAs) with different carbon chain length. In male rats, perfluoroheptanoic acid (PFHA) was rapidly eliminated in urine with the proportion of 92% of the dose being eliminated within 120 h after an intraperitoneal injection. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) was eliminated in urine with the proportions of 55, 2.0 and 0.2% of the dose, respectively. By contrast, four PFCAs were eliminated in feces with the proportion of less than 5% of the dose within 120 h after an injection. In female rats, the proportions of PFOA and PFNA eliminated in urine within 120 h were 80% and 51% of the dose, respectively, which were significantly higher compared with those in male rats. There was the tendency that PFCA with longer carbon chain length is less eliminated in urine in both male and female rats. Fecal elimination of PFCAs was not different between PFCAs in female rats and comparable to those in male rats. The rates of biliary excretion of PFCAs in male rats were slower than those in female rats. Sex-related difference in urinary elimination of PFOA was abolished when male rats had been castrated. On the contrary, treatment with testosterone suppressed the elimination of PFOA in urine in both castrated male rats and female rats. The effect of testosterone was in a time- and dose-dependent manner. These results suggest that PFCAs are distinguished by their carbon chain length by a renal excretion system, which is regulated by testosterone.     

Evidence contradicting the notion that gonadal hormones regulate brain opiate receptors

Castration of male rats has been reported to increase brain opiate receptors by nearly 100%. We assayed brain opiate receptors with both naloxone and met-enkephalin, but found no effect of gonadectomy on the Kd or Bmax for either ligand in male or female mice or in male rats. Experiments were performed with 2 strains of mice and 3 strains of rats; mice were gonadectomized 1–7 weeks and rats were castrated 3 weeks before assay. Both washed and unwashed brain membrane preparations were used. Administration of testosterone or estrogen to intact male or female mice did not alter opiate receptors. Castration did not affect the strain or age and brain-region differences found for naloxone binding in male rats.     

Flutamide blocks the self-priming effect of luteinizing hormone-releasing hormone in pubertal male rats

Castration of pubertal or young adult male rats eliminates the self-priming effect of luteinizing hormone-releasing hormone on luteinizing hormone secretion. Testosterone, dihydrotestosterone, or estradiol will maintain this effect in castrated animals. In order to explore the mechanism by which both dihydrotestosterone and estradiol are capable of maintaining the effect, intact rats as well as castrated animals implanted with testosterone capsules were treated with the antiandrogen Flutamide. In both intact animals and castrated rats bearing testosterone-filled Silastic capsules, Flutamide blocked the self-priming effect. These data suggest that the androgen receptor is of primary importance in the maintenance of the self-priming effect.     

Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats

The potency of the induction of peroxisomal beta-oxidation was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats. In male rats, perfluoroheptanoic acid (PFHA) has little effect, although perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) potentially induced the activity. By contrast, PFHA and PFOA did not induce the activity of peroxisomal beta-oxidation in the liver of female rats while PFNA and PFDA effectively induced the activity. The induction of the activity by these PFCAs was in a dose-dependent manner, and there is a highly significant correlation between the induction and hepatic concentrations of PFCAs in the liver regardless of their carbon chain lengths. These results strongly suggest that the difference in their chemical structure is not the cause of the difference in the potency of the induction. Hepatic concentrations of PFOA and PFNA was markedly higher in male compared with female rats. Castration of male rats reduced the concentration of PFNA in the liver and treatment with testosterone entirely restored the reduction. In contrast to the results obtained from the in vivo experiments, the activity of peroxisomal beta-oxidation was induced by PFDA and PFOA to the same extent in cultured hepatocytes prepared from both male and female rats. These results, taken together, indicate that difference in accumulation between PFCAs in the liver was responsible for the different potency of the induction of peroxisomal beta-oxidation between PFCAs with different carbon chain lengths and between sexes.     

Testosterone and GHRH gene expression in adult and old rats

The effect of castration and testosterone replacement on GHRH gene expression was evaluated in adult male rats. Castration for 21 days did not affect GHRH mRNA levels, and also ineffective in this context was acute or chronic administration of testosterone to castrated rats. Hypothalamic GHRH mRNA was significantly reduced in aged male rats, but restoration of plasma testosterone concentrations via implanted capsules did not modify the low levels of GHRH mRNA. All in all, these findings support the notion that in adult and aged male rats GHRH-producing structures are insensitive to androgens.     

The SUMO-Specific Protease Senp2 Regulates SUMOylation,Expression and Function of Human Organic Anion Transporter 3

Organic anion transporter 3 (OAT3) plays a vital role in removing a broad array of anionic drugs from kidney, thereby avoiding their possibly toxic side effects in the body. We earlier demonstrated that OAT3 is subjected to a specific type of post-translational modification called SUMOylation. SUMOylation is a dynamic event, where de-SUMOylation is catalyzed by a class of SUMO-specific proteases. In the present investigation, we assessed the role of SUMO-specific protease Senp2 in OAT3 SUMOylation, expression and function. We report here that overexpression of Senp2 in COS-7 cells led to a reduced OAT3 SUMOylation, which correlated well with a decreased OAT3 expression and transport activity. Such phenomenon was not observed in cells overexpressing an inactive mutant of Senp2. Furthermore, transfection of cells with Senp2-specific siRNA to knockdown the endogenous Senp2 resulted in an increased OAT3 SUMOylation, which correlated well with an enhanced OAT3 expression and transport activity. Coimmunoprecipitation experiments showed that Senp2 directly interacted with OAT3 in the kidneys of rats. Together these results provided first demonstration that Senp2 is a significant regulator for OAT3-mediated organic anion/drug transport.     

Renal elimination of organic anions in rats with bilateral ureteral obstruction

Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.     

Decreased renal organic anion secretion and plasma accumulation of endogenous organic anions in OAT1 knock-out mice

The "classical" organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of approximately 60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.     

Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

Effects of feminization of male F344 rats on induction of tumors and on nucleic acid alkylation by nitrosobis-(2-oxopropyl)amine

Groups of male and female F344 rats were treated twice weekly by gavage with 2.5 mg of nitrosobis-(2-oxopropyl)amine (BOP) for 35 weeks. Additional groups given the same treatment were male rats castrated at birth, male rats bearing an implant of a pellet containing estradiol and castrated male rats bearing an estradiol pellet. Most rats died with tumors related to the treatment; intact male rats survived the least well of the five groups. Most rats in all groups had alveolar/bronchiolar neoplasms of the lung. Many of the male rats also had follicular cell neoplasms of the thyroid and transitional cell neoplasms of the urinary bladder and kidney pelvis; there were no liver tumors in intact male rats. Almost all female rats and castrated male rats had liver neoplasms, including hepatocellular, cholangiocellular and hemangiosarcomatous neoplasms, but few neoplasms of the thyroid, kidney or bladder. The male rats feminized with estradiol, intact or castrated, had liver neoplasms, mainly cholangiocellular, and also neoplasms of the thyroid. Two rats of each of the five groups were treated at 20 weeks of age with [14C]BOP. As measured by respiration of 14CO2, metabolism of BOP was faster in the two groups of male rats with the estradiol implant than in the other groups. DNA and RNA of the liver were isolated 6 h after treatment. The extent of methylation of liver DNA as 7-methylguanine and O6-methylguanine was higher in the females and in the feminized males than in the intact male rats, but when normalized to the dose of nitrosamine per unit body weight there was little difference among the five groups.