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Endocrinology Vol. 144, No. 6 2659-2668
Copyright © 2003 by The Endocrine Society

Human Uridine Diphosphate-Glucuronosyltransferase UGT2B7 Conjugates Mineralocorticoid and Glucocorticoid Metabolites

Caroline Girard, Olivier Barbier, Guy Veilleux, Mohamed El-Alfy and Alain Bélanger

Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (Centre Hospitalier de l’Université Laval) and Laval University, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Alain Bélanger, Ph.D., Oncology and Molecular Endocrinology Research Center, 2705 Boulevard Laurier, Sainte-Foy, Québec, Canada G1V 4G2. E-mail: alain.belanger{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mineralocorticoid and glucocorticoid hormones are metabolized as glucuronide conjugates. Using labeled [14C]uridine diphosphate glucuronic acid and microsomal preparations from human embryonic kidney 293 cells stably expressing the different human and monkey uridine diphosphate glucuronosyltransferase (UGT)2B enzymes, it is demonstrated that the two human allelic variants UGT2B7H(268) and UGT2B7Y(268) conjugate aldosterone, its A-ring reduced metabolites (5{alpha}-dihydroaldosterone and 3{alpha},5ß-tetrahydroaldosterone), and both 5{alpha}- and 5ß-tetrahydrocortisone epimers. The two variants of UGT2B4 also glucuronidate tetrahydroaldosterone, whereas all enzymes tested were inefficient to produce cortisol glucuronide derivatives. Kinetic analyses reveal that UGT2B7 polymorphisms glucuronidate mineralocorticoids with a 5.5- to 20-fold higher affinity than glucocorticoids. For the first time, a significant difference between the two allelic variants of UGT2B7 is described, because UGT2B7H(268) possesses an 11-fold higher aldosterone glucuronidation efficiency (ratio Vmax(app.)/Km(app.)) than UGT2B7Y(268). RT-PCR experiments demonstrate the expression of UGT2B7 in human kidney and in renal proximal tubule epithelial cells, suggesting that mineralocorticoids and glucocorticoids are metabolized in their target tissue. Measurement of aldosterone glucuronidation and normalization with the UGT2B protein contents in monkey tissues demonstrate that liver and kidney glucuronidate this hormone with a similar velocity. Immunohistochemical studies performed in monkey kidney cortex reveal a restrictive expression of UGT2B proteins in the epithelial cells of the proximal tubules. Because expression of the mineralocorticoid receptor was detected in the distal tubule epithelial cells, the present data suggest a two-cell mechanism of aldosterone action and metabolism in the kidney.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN ADRENALS secrete a variety of steroids including aldosterone, the major mineralocorticoid that regulates plasmatic electrolyte homeostasis; cortisol, which controls cellular metabolism and resistance to stress factors; and, finally, dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), the precursors of androgens (1, 2, 3). In recent years, the catabolism of androgens has been extensively investigated, and the formation of androgen glucuronide was found as a major pathway for the elimination of these hormones (4). Several studies also indicate that glucuronidation of aldosterone and cortisol and their metabolites would be an inactivating and metabolic pathway for such steroids (5, 6).

In a first step of the catabolic process, active mineralocorticoids and glucocorticoids are reduced to dihydro and tetrahydro derivatives in the liver (3). Subsequently, the reduced metabolites are conjugated with glucuronic acid in both the hepatic and renal parenchymas (3). The importance of aldosterone hepatic reduction is such that up to 90% of the circulating free aldosterone is removed after one passage through the liver, and its half-life is only 20–35 min (7). However, via an enterohepatic cycle, a large proportion of aldosterone is reabsorbed due to deconjugation by enzymes of the intestinal bacteria (8). Nevertheless, aldosterone-18-glucuronide and tetrahydroaldosterone (THA)-glucuronide measured in urine reflect 5–15% and 15–40% of the daily aldosterone secretion, respectively (5, 9). As for aldosterone, glucuronidation is an important pathway of the metabolism of cortisol, which has a half-life of 80–120 min in the circulation. More than 91% of cortisol metabolites found in human urine correspond to conjugate derivatives, and mainly to glucuronides (6, 10).

In mammals, a huge variety of endogenous and exogenous compounds are metabolized through glucuronidation. This reaction, which corresponds to the transfer of the glucuronosyl group from uridine diphosphate (UDP)-glucuronic acid (UDPGA) to small hydrophobic molecules, is catalyzed by members of the UDP-glucuronosyltransferase (UGT) enzyme super-family (11). Based on the homology of primary structures, the UGT proteins have been categorized into two major families, UGT1 and UGT2, with the UGT2 further divided into two sub-families, UGT2A and UGT2B (11). In humans, seven UGT2B enzymes have been cloned and characterized (UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28; Refs. 12, 13, 14, 15, 16, 17, 18). These proteins are encoded by different genes clustered on chromosome 4q13–4q21.1 and catalyze preferentially the glucuronidation of endogenous molecules such as steroids (19, 20, 21), with the exception of UGT2B10 and UGT2B11, which are considered as orphan enzymes (17, 18).

Although the liver is a major conjugating organ, UGT enzymes are also expressed in a variety of extrahepatic tissues, including kidney, steroid target tissues, and intestine (4, 17, 22). Analysis of the glucuronidation activity of more that 20 tissues in the monkey demonstrated that steroid target tissues, such as ovary, seminal vesicle, and prostate highly glucuronidate steroid hormones and their metabolites (23). Furthermore, these enzymes are expressed in a cell-type specific manner in the rat, human, and monkey, as shown by several immunohistochemical studies (24, 25, 26).

Although the conjugation of glucuronic acid to aldosterone, cortisol, and their metabolites has been known for many years, the UGT enzyme(s) that catalyze this reaction have not been identified until now. In the present study, we analyze the conjugation of these compounds by human and monkey UGT2B enzymes. The cynomolgus monkey (Macaca fascicularis) is particular in having elevated concentrations of glucuronidated androgen metabolites in the circulation, and the presence of UGT enzymes in several tissues other than the liver is well established in this species (25, 27, 28). The kinetic parameters of mineralocorticoid and glucocorticoid substrates for the major UGT enzymes involved are reported. Immunohistological analyses of the monkey kidney with an anti-UGT2B antibody demonstrate specific localization of these proteins in epithelial cells of the convoluted tubules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Experiments were performed at the animal facilities of the Centre de Recherche du Centre Hospitalier de l’Université Laval (CHUL; Québec, Canada). Animals were housed individually or in pairs in standard stainless cages in a room maintained at 23 ± 3 C with a 12-h light, 12-h dark cycle (lights on at 0715 h). Animals were fed four primate cookies twice daily at a 6-h (± 1h) interval. Fruits and/or vegetables were distributed twice weekly. Water was available ad libitum. Throughout this study, animals were maintained and handled in accordance with the written policies of the Canadian Council on Animal Care and the NIH Guide for the Care and Use of Laboratory Animals (7th ed., 1996). This study has been approved by the ethical committee for animal protection of CHUL Research Center. For protein and mRNA analyses, an adult male was injected with ketamine and removed from its cage, before inducing rapid anesthesia with saffan (1.2 mg/kg) and ketamine (10 mg/kg; 1:1 vol/vol). After euthanasia, tissues were collected and freed from fat and connective tissues immediately before freezing in liquid nitrogen.

Materials
UDPGA and cortisol were obtained from Sigma (St. Louis, MO). Other aglycons were purchased from Steraloids Inc. (Wilton, NH). [14C]UDPGA (285 mCi/mmol) was obtained from NEN Life Science Products (Boston, MA). Human liver and kidney microsome preparations were purchased from the Human Cell Culture Center, Inc. (Laurel, MD). A kit for RT-PCR including Superscript II was provided by Life Technologies, Inc. (Rockville, MD), and Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). Human embryonic kidney 293 (HK293) cells were obtained from the American Type Culture Collection (Rockville, MD). Renal proximal tubule epithelial cells (RPTEC) and related media were from Clonetics (Walkersville, MD). The isolation of human and monkey UGT2B cDNAs and their stable expression in HK293 cells have been described previously (12, 13, 14, 15, 16, 27, 28, 29, 30, 31).

Isolation of microsomal proteins from monkey tissues and from RPTEC, HK293, and UGT-expressing HK293 cells
RPTEC, untransfected HK293, and HK293 cells stably expressing each human and monkey UGT2B isoform were harvested, frozen twice in liquid nitrogen, and homogenized in K2HPO4 0.1 M, KH2PO4 0.1 M (pH 7.4), glycerol 20%, EDTA 1 mM, dithiothreitol 1 mM, 2.5 µg/ml pepstatin, and 0.5 µg/ml leupeptin using a Potter-Glas-col (Terre Haute, IN) type homogenizer with a Teflon pestle at 4 C. Monkey liver, kidney, adrenals, heart, brain, skin, and salivary gland were homogenized in the same conditions as cultured cells. Cell and tissue homogenates were centrifuged at 12,000 x g, 4 C for 20 min. Supernatants were centrifuged at 105,000 x g for 1 h at 4 C. Microsome pellets were resuspended in 0.2 ml of homogenization buffer, and protein contents were determined using the Bradford’s reagent, with BSA for standard curves (32). Samples were aliquoted and kept at -80 C until Western blot analyses or glucuronidation assays.

Western blot analyses
Microsomal proteins (15 µg) from monkey liver and kidney or from RPTEC, untransfected HK293, or HK293-UGT2B7H(268) cells were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel. After migration, the gel was transferred onto a nitrocellulose membrane (Xymotech, Montréal, Québec, Canada) and hybridized with the EL-93 antibody (a specific anti-UGT2B antibody; Ref. 33). An antirabbit IgG antibody conjugated with peroxidase (Amersham, Québec, Canada) was used as the second antibody, and the resulting immunocomplexes were visualized using the Western blot Chemiluminescence Reagent Plus as specified by the manufacturer (NEN Life Science Products) and quantified by BioImage Visage 110s (Genomic Solution Inc., Ann Arbor, MI).

Glucuronidation assays on aldosterone, cortisol, and their metabolites
To screen for enzymes or tissues that catalyze the glucuronidation of aldosterone, 5{alpha}-dihydroaldosterone (DHA), 3{alpha},5ß-THA, cortisol, 5{alpha}-tetrahydrocortisone (THE), and 5ß-THE, microsomes (20 µg) from human and monkey tissues, and from RPTEC, HK293, and HK293 cells expressing all human and monkey UGT2B isoforms were incubated with these substrates (200 µM) in the presence of 7.5 µM [14C]UDPGA and 92.5 µM unlabeled UDPGA. Assays were performed in 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 8.5 mM saccharolactone, 10 µg/ml phosphatidylcholine, 2.5 µg/ml pepstatin, and 0.5 µg/ml leupeptin for 16 h at 37 C and were terminated by adding 100 µl of methanol. Products were subsequently loaded on thin layer chromatography plates and migrated in a toluene-methanol-acetic acid (7:3:1 ratio) solvent for 2 h. Compounds and proteins that demonstrated reactivity in this screening were reassayed in a quantitative experiment in the same buffer containing 7.5 µM [14C]UDPGA, 492.5 µM unlabeled UDPGA, and 200 µM aglycon for 4 h at 37 C. Glucuronide product formation was quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). As previously reported (12, 13, 14, 15, 16, 27, 28, 29, 30, 31), 500 µM UDPGA and 4 h represent saturating and linear conditions of glucuronidation catalyzed by UGT2B enzymes.

Liquid chromatography coupled with mass spectrometry (LC-MS/MS)
Because no cortisol-glucuronides were detected in classical glucuronidation assays, microsomal proteins (20 µg) from human and monkey tissues and from HK293 cell lines expressing all human and monkey UGT2B isoforms were incubated with cortisol in the same conditions as for screening in the presence of 500 µM of unlabeled UDPGA and in the absence of radiolabeled UDPGA. Assays were terminated by adding 100 µl of methanol and centrifuged at 13,000 x g for 5 min to remove precipitated proteins. Subsequently, 25 µl of the supernatant were analyzed by LC-MS/MS without extraction. Compounds were separated by HPLC using a zorbax C18 column (7.5 x 4.6 mm), and elution was performed with a gradient of methanol-ammonium formate (1 mM), from 25% in water-ammonium formate (1 mM) to 95% in 10 min (0.8 ml*min-1). Mass spectrometry, used to determine the molecular weight of compounds isolated by chromatography, was conducted using a full scan approach with mass specter from 200–650 m/z. Detection of conjugated compounds was performed by using an Ion-Trap Mass Spectrometer, LCQ detector in negative electrospray ionization mode (Finnigan, San Jose, CA).

Determination of apparent Km [Km(app.)] and apparent maximal velocity [Vmax(app.)] of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, and 5ß-THE glucuronidation by human and monkey enzymes
Kinetic analyses were performed for 4 h using microsomal proteins of HK293 cells stably expressing UGT2B7H(268), UGT2B7Y(458), UGT2B18, UGT2B19, or UGT2B30. Microsomes were incubated with 7.5 µM [14C]UDPGA, 492.5 µM unlabeled UDPGA, and substrate concentrations ranging from 0.1–500 µM in the same mix as glucuronidation assays. The Km(app.) and Vmax(app.) values, obtained by double reciprocal plots (Lineweaver-Burk), represent the mean of three independent experiments performed in duplicate. The SD values were determined on the mean of three separate experiments.

RT-PCR analyses
The expression of UGT2B4, UGT2B7, the mineralocorticoid or glucocorticoid receptors (MR and GR, respectively) in human liver and kidney cells, and RPTEC was analyzed by RT-PCR. The RT reactions were performed using 1.5 µg of total RNA from each tissue or cell line in the presence of 200 U of Superscript II in a mix containing 200 µM of deoxynucleotide triphosphates, 1x First-Strand buffer, dithiothreitol, and 500 pmol/µl of an antisense primer specific for UGT2B4 (5'-CTTCTTCATGTCAAATATTTGGAACCAAAAT-3'), UGT2B7 (5'-CTGATCCCACTTCTTCATGTCAA ATATTTC-3'), MR (5'-AAAGTAAAGGATTTATTGTAATCTG-3'), or GR (5'-GAATTCAATACTCATGGTCTTATCC-3'), and of 2 pmol of the antisense glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer (5'-AGCCCCAGCGTCAAAGGTGG-3') in a total volume of 20 µl for 90 min at 37 C. All PCRs were performed with one tenth of the RT products in a 100-µl PCR containing 200 µM of deoxynucleoside triphosphates, 1x cloned Pfu DNA polymerase buffer, 5 U of the PfuTurbo DNA Polymerase for 35 cycles (45 sec at 94 C, 45 sec at 54 C, 1 min at 72 C), after which 10 µl of PCR products were loaded on a 1% ethidium bromide-stained gel. The PCRs for UGT2B4, UGT2B7, and GAPDH used 100 pmol of the specific sense primer (UGT2B4, 5'-GAAATGGACTTCAGCTCTTCTGCTGATC-3'; UGT2B7, 5'-CAAGGATGTCTGTGAAATGGACTTCAGTAA-3'; GAPDH, 5'-CATGTTCGTCATGGGTGTGAACCATGAG-3') and 100 pmol of the corresponding antisense primer described above. MR was amplified with 100 pmol of the sense primer 5'-CAAACAGATGATCCAAGTCGTGAAG-3' and of the antisense primer 5'-TCCAAACCTCTGACATGACTTAAAC-3'. Sense and antisense primers for GR amplification were 5'-CGGTGTGCTCTGATGAAGCTTCAGG-3' and 5'-CACCAACAGTGACACCAGGGTAGG-3', respectively.

Tissue preparation and immunohistochemical localization of UGT2B proteins
For immunohistochemistry experiments, a male monkey was perfused in the heart with PBS at 25 C, followed by 4% paraformaldehyde in phosphate buffer (0.05 M) for 30 min. Tissues (including kidney) were then post-fixed in 4% paraformaldehyde and phosphate buffer (0.05 M) 4 C for 24 h, processed, and embedded in paraffin. Paraffin sections (4 µm) were cut, mounted, deparaffinized in toluene, and then rehydrated. Immunostaining was performed using the anti-UGT2B EL-93 antisera diluted 1:300 in Tris saline (pH 7.6) for 1 h at room temperature, as previously described (26). After incubation, sections were washed with PBS and incubated with a biotin-labeled goat antirabbit {gamma}-globulin diluted 1:1500 for 10 min. Then, the sections were treated with streptavidin coupled with peroxidase, and diaminobenzidine was used as the chromogen to visualize the biotin-streptavidin-peroxidase complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA). Endogenous peroxidase activity was eliminated by preincubation with 3% H2O2 for 20 min. The intensity of the staining was controlled under the microscope. The sections were then counterstained with hematoxylin. Control experiments were performed on adjacent sections by substituting preimmune rabbit serum (1/100).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glucuronidation of mineralocorticoid and glucocorticoid metabolites by stably expressed human UGT2B enzymes
A screening assay was performed in which microsomal proteins from HK293 cell lines stably expressing UGT2B4D(458), UGT2B4E(458), UGT2B7H(268), UGT2B7Y(268), UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28 were incubated with aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, cortisol, 5{alpha}-THE, and 5ß-THE. UGT2B7H(268) and UGT2B7Y(268) catalyzed the transfer of glucuronic acid to aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, 5{alpha}-THE, and 5ß-THE, whereas the two allelic variants of UGT2B4 only conjugated 3{alpha},5ß-THA (Fig. 1Go). Other human UGT2B enzymes were not reactive with these substrates, and glucuronidation of cortisol was not detected in both classical glucuronidation assays with radioactive UDPGA and LC-MS/MS system (data not shown). After this screening assay, reactive substrates were assayed in a quantitative analysis in the presence of UGT2B4D(458), UGT2B4E(458), UGT2B7H(268), and UGT2B7Y(268) proteins. UGT2B7H(268) showed a 8.9-, 6.2-, 3.8-, and 2.0-fold higher activity than UGT2B7Y(268) for glucuronidation of aldosterone, 5ß-THE, 5{alpha}-THE, and 3{alpha},5ß-THA, respectively. By contrast, the two allelic variants of UGT2B7 glucuronidated 5{alpha}-DHA at the same level, and UGT2B4D(458) and UGT2B4E(458) shared similar 3{alpha},5ß-THA conjugation activities (Fig. 2Go).



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  		Figure 1. Glucuronidation of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, 5{alpha}-THE, and 5ß-THE by human UGT2B proteins analyzed by thin layer chromatography. Glucuronidation of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, 5{alpha}-THE, and 5ß-THE by human liver, kidney, and HK293 cells stably expressing all human UGT2B enzymes, was assessed by incubation of microsomal proteins with 7.5 µM [14C]UDPGA and 92.5 µM unlabeled UDPGA and chromatographed with thin layer plate. The free UDPGA is detected at the bottom of the chromatogram.

 


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  		Figure 2. Reactivity of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, 5{alpha}-THE, and 5ß-THE with the two UGT2B7 and UGT2B4 polymorphic enzymes. Glucuronidation activity values were determined using microsomal proteins from UGT2B-HK293 cell lines incubated in the presence of 200 µM of aglycons, 7.5 µM [14C]UDPGA, and 492.5 µM unlabeled UDPGA for 4 h at 37 C. Values represent the mean (±SD) of three independent experiments, each performed in duplicate.

 
Kinetic analyses performed with microsomal proteins revealed that the two allelic variants of UGT2B7 possess Km(app.) values in the micromolar range for all substrates (Table 1Go). However, both enzymes presented lower Km(app.) values for mineralocorticoids (between 0.23 and 4.70 µM) than for 5ß-THE [64.57 and 64.68 µM for UGT2B7H(268) and UGT2B7Y(268), respectively]. Interestingly, UGT2B7Y(268) displayed a 2-fold lower Km(app.) value compared with UGT2B7H(268) for glucuronidation of aldosterone and THA, thus indicating a higher affinity of the UGT2B7Y(268) variant for these substrates. In contrast, 5{alpha}-DHA was glucuronidated with a higher affinity by UGT2B7H(268), whereas both polymorphic enzymes shared a similar affinity for 5ß-THE. Vmax(app.) for glucuronidation varied from 4.8–177.5 pmol*min-1*mg protein-1 for aldosterone glucuronidation by UGT2B7Y(268) and 5ß-THE by UGT2B7H(268), respectively. Because the stable HK293-UGT2B7H(268) cell line expressed 2.5-fold more UGT proteins than HK293-UGT2B7Y(268) cells (12, 34), the glucuronidation efficiency values were adjusted for protein contents (Table 1Go). After this correction, an 11-fold higher aldosterone glucuronidation efficiency was observed for UGT2B7H(268) compared with UGT2B7Y(268), indicating that the single amino acid change greatly affects the efficiency of mineralocorticoid conjugation by UGT2B7. By contrast, the normalized glucuronidation efficiencies of both proteins were more similar for other substrates (Table 1Go).


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  		Table 1. Kinetic parameters of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, and 5ß-THE glucuronidation by the two allelic variants of UGT2B7

 
Expression of UGT2B transcripts and proteins in human liver and kidney and the RPTEC
As previously reported (22), RT-PCR analyses revealed that both UGT2B4 and UGT2B7 are expressed in human liver and kidney (Fig. 3AGo), whereas HK293 cells used as negative control did not express any UGT2B enzymes (data not shown). Interestingly, UGT2B4 and UGT2B7 mRNAs were also detected in RPTEC (Fig. 3AGo). Furthermore, microsomal proteins purified from RPTEC, untransfected HK293 cells, and HK293-UGT2B7H(268) cells were immunoblotted with the anti-UGT2B antibody. UGT2B proteins were detected in both RPTEC and HK293-UGT2B7 cells but not in untransfected HK293 cells (Fig. 3CGo). To evaluate the potential action of aldosterone and cortisol hormones in RPTEC, the expression of MR and GR mRNAs was analyzed by RT-PCR (Fig. 3BGo). Expression of both receptors was detected in human kidney control, as expected (1, 2), whereas in RPTEC only GR transcripts were amplified.



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  		Figure 3. Expression of UGT2B4, UGT2B7, mineralocorticoid and glucocorticoid receptors in human liver and kidney and in RPTEC. The expression of UGT2B4 and UGT2B7 (A) and mineralocorticoid and glucocorticoid receptor (B) transcripts in total RNA samples of human liver and kidney cells and RPTEC was assessed by RT-PCR analyses using specific oligonucleotides. The integrity of each RNA preparation was verified by amplification of the GAPDH transcript. C, The presence of UGT2B proteins in microsomal preparation (15 µg) of RPTEC was demonstrated by immunoblot analyses using the anti-UGT2B EL-93 antibody. The 53-kDa UGT2B protein is observed in these cells, as well as in microsomes from HK293-UGT2B7H(268) cells, as a positive control. As expected, no UGT2B protein was observed in microsomes from untransfected HK293 cells.

 
Glucuronidation of mineralocorticoids and glucocorticoids by monkey UGT2B enzymes
Monkey UGT2B isoforms were also screened to identify UGT2B enzymes catalyzing the conjugation of glucocorticoids and mineralocorticoids (Fig. 4Go). Microsomes from monkey liver and kidney were used as control, and, as expected, glucuronide derivatives of all substrates tested were formed, except for cortisol. Aldosterone was specifically conjugated by UGT2B19, whereas 5{alpha}-DHA glucuronidation was observed with both UGT2B18 and UGT2B19. UGT2B18 was the major 3{alpha},5ß-THA conjugating enzyme, but a low level of 3{alpha},5ß-THA glucuronidation was observed for UGT2B9, UGT2B20, and UGT2B23 (Fig. 4Go). Interestingly, 5{alpha}-THE and 5ß-THE glucuronides were detected only when corresponding aglycons were incubated with microsomes from HK293-UGT2B30 cells. As observed for human UGT2B enzymes, monkey UGT2Bs possessed no significant glucuronidation activity toward cortisol (data not shown). In quantitative analyses, the enzyme with the highest conjugating activity was UGT2B18 for 3{alpha},5ß-THA (146.58 pmol*min-1*mg protein-1; Fig. 5Go). UGT2B19 was 3-fold more active for 5{alpha}-DHA than aldosterone. Interestingly, 5{alpha}- and 5ß-THE epimers were glucuronidated at the same level by UGT2B30, indicating that this enzyme has no stereospecificity for reduced glucocorticoids (Fig. 5Go).



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  		Figure 4. Glucuronidation of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, 5{alpha}-THE, and 5ß-THE by cynomolgus monkey UGT2B enzymes. Microsomes from monkey liver and kidney tissues, and from UGT2B9, UGT2B18, UGT2B19, UGT2B20, UGT2B23, and UGT2B30-HK293 cells were analyzed by thin layer chromatography for the conjugation of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA, 5{alpha}-THE, and 5ß-THE.

 


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  		Figure 5. Glucuronidation activities catalyzed by monkey UGT2B18, UGT2B19, and UGT2B30 enzymes. Glucuronidation activity values were determined as described in Materials and Methods. Values represent the mean (± SD) of three independent experiments, each performed in duplicate.

 
Monkey UGT2B enzymes showed Km(app.) values in the micromolar range for mineralocorticoid and glucocorticoid substrates (Table 2Go). Interestingly, the Km(app.) of aldosterone and DHA glucuronidation by UGT2B19 and UGT2B18 enzymes were lower when compared with the Km(app.) of UGT2B30 and UGT2B18 for 5ß-THE and 3{alpha},5ß-THA conjugation, respectively. Km(app.) values of human and monkey enzymes correlated with the level of aldosterone reduction (lower for aldosterone and higher for THA), indicating that the presence of a hydroxyl group on the aldosterone molecule decreases its affinity for UGT enzymes. Furthermore, because the Vmax(app.)/Km(app.) ratio of UGT2B18 glucuronidation varied from 35.4–3.7 µl*min-1*mg protein-1 for DHA and THA, respectively (Table 2Go), the decreased affinity of hydroxylated substrates is associated with a lower glucuronidation efficiency.


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  		Table 2. Kinetic parameters of the glucuronidation of aldosterone, 5{alpha}-DHA, 3{alpha},5ß-THA and 5ß-THE by cynomolgus monkey UGT2B enzymes

 
Glucuronidation of aldosterone by monkey tissues
The tissue distribution of aldosterone conjugation was analyzed by measuring the formation of aldosterone glucuronide in liver, kidney, adrenals, heart, brain, salivary glands, and skin (Fig. 6AGo). The highest activity was detected in liver and kidney. However, Western blot experiments showed that the liver expresses approximately three times more of both glycosylated and unglycosylated UGT2B proteins than the kidney (Fig. 6BGo), and adjusting the aldosterone-glucuronide formation with the UGT2B protein contents in the corresponding tissue led to similar conjugation activities in liver and kidney (Fig. 6Go).



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  		Figure 6. Levels of aldosterone glucuronidation activity in tissues of the cynomolgus monkey. A, The aldosterone conjugating activity in each tissue was determined using microsomal proteins (white bars), and liver and kidney conjugating values were normalized (black bars) by the level of UGT2B protein expression, as determined by immunoblot analysis (B).

 
Localization of UGT2B proteins in monkey kidney cortex
Using the anti-UGT2B EL-93 antibody (25, 33), an immunohistochemical study performed on sections from the cortical region of male monkey kidney revealed that the expression of UGT2B enzymes is restricted to epithelial cells of proximal tubules (Fig. 7Go, A and B). These cells were strongly stained, whereas no labeling was observed in glomerulus. A weak labeling was observed in some epithelial cells of collecting ducts and in endothelial cells of blood vessels (Fig. 7BGo). When the antibody was replaced by the preimmune serum, a total absence of labeling confirmed the specificity of immunostaining (Fig. 7Go, A and C).



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  		Figure 7. Cell type-specific expression of UGT2B proteins in the monkey kidney cortex determined by immunohistochemistry with the anti-UGT2B antibody (1:300). A, Expression of UGT2B proteins is detected in epithelial cells of proximal tubules (arrows), whereas no labeling is observed in the glomerulus (G) or in the distal tubules. B, An intense immunostaining reaction is observed in epithelial cells of the proximal tubules (P), whereas some epithelial cells of the collecting ducts (C) and of capillaries (A) were significantly stained. C and D, Similar sections as in A and B, respectively, where the antibody was replaced by the preimmune serum. No immunostaining reaction could be detected. Magnification, A and C, x100; B and D, x250.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cortisol is metabolized by 5{alpha}- and 5ß-reductases to hydroxycortisone and by reversible interconversion to inactive cortisone (35, 36). In human urine, 91.7% of cortisol metabolites are excreted as conjugated derivatives, and almost 90% of these conjugates correspond to monoglucuronides (6). Of these glucuronidated compounds, less than 5% correspond to 4-en-3-oxosteroids derivatives (cortisol, cortisone, and 20-dihydrocortisol), whereas the major glucuronide is 5ß-THE glucuronide (6). Thus, the absence of cortisol glucuronidation observed in human tissues (liver and kidney) and stably expressed UGT2B enzymes is consistent with the very low concentration of cortisol-glucuronide found in human urine (6). In human and monkey, tetrahydroxylated cortisone (THE) metabolites are conjugated specifically by UGT2B7 and UGT2B30, respectively. Human UGT2B7 is encoded by a polymorphic gene, and the two variants UGT2B7H(268) and UGT2B7Y(268) are able to glucuronidate A-ring reduced metabolites of cortisol. Kinetic analyses revealed that the two allelic variants of UGT2B7 possess the same affinity for 5ß-THE, whereas efficiency value as determined by the ratio Vmax(app.)/Km(app.) is higher for UGT2B7H(268).

The most impressive difference between the two allelic variants of the UGT2B7 enzyme concerns the efficiency of aldosterone glucuronidation. Despite only a 2-fold higher Km(app.) value, UGT2B7H(268) is 11 times more efficient than UGT2B7Y(268) for the glucuronidation of this steroid hormone. This result clearly demonstrates that a single mutation in the UGT2B7 gene may affect the conjugating activity of the corresponding protein. This observation further reinforces the importance of single residues within the UGT2B proteins for the integrity of their enzymatic activities, as previously reported (37). Among human UGT2B enzymes, UGT2B7 possesses the largest range of substrate specificity and conjugates a huge variety of endogenous or exogenous compounds such as estrogens (38, 39, 40), androgens (38, 40), fatty acids (41, 42), retinoids (43, 44), bile acids (38), and exogenous aglycons (for review, see Ref. 45). However, the effect of the allelic variation H/Y(268) on the glucuronidation activity of UGT2B7 was not so elevated as for aldosterone conjugation (12, 39). Interestingly, several studies report a 2- to 10-fold higher prevalence of the UGT2B7H(268) polymorphism in the Asian population, whereas variant alleles are equally distributed in Caucasian populations (46, 47). Considering the difference in aldosterone glucuronidation by the two polymorphic enzymes, it would be interesting to compare the urinary level of aldosterone-18-glucuronide in patients within Asian populations, with respect to their UGT2B7 polymorphic expression.

The major difference between the aldosterone glucuronidation efficiency values of the two UGT2B7 variants suggests that this single nucleotide polymorphism affects the catalytic domain of the enzyme. However, the unique substrate binding site of UGT2B7 for steroids and xenobiotics was localized between residues 72 and 168 (48, 49, 50, 51, 52). Because the amino acid change introduced by the single nucleotide polymorphism is located at 100 amino acids of the substrate binding site, this suggests that the 11-fold higher efficiency of aldosterone glucuronidation by UGT2B7H(268) is not due to a modification of the aglycone binding site of this enzyme. However, it remains possible that the bulky C21-steroid structure of aldosterone requires a larger or an additional binding site on UGT2B7. A similar mechanism was proposed for the binding of the UDPGA cofactor onto UGT proteins because it was suggested that UGT proteins are folded in such a manner that UDPGA spans the carboxyl- and amino-terminal domains of the enzyme (49, 51, 53).

In monkey, aldosterone is conjugated specifically by UGT2B19, whereas 5{alpha}-DHA glucuronidation is catalyzed by UGT2B18 and UGT2B19, and only UGT2B18 conjugated 3{alpha},5ß-THA. These results demonstrate a significant difference between human and monkey in the mineralocorticoid glucuronidation because two monkey enzymes are required to metabolize the three substrates, whereas in human, UGT2B7 was capable of conjugating the three substrates and UGT2B4 had only low activity on 3{alpha},5ß-THA.

Analysis of aldosterone glucuronidation in different monkey tissues revealed that the hormone is mainly metabolized in liver and kidney. These results, are consistent with different studies reporting that the liver is the major site of aldosterone metabolism in rat, dog, and human, and that the kidney also conjugates this hormone (54, 55). Using classical UGT substrates, glucuronidation activity measurement showed that following the liver, the highest UGT activity is found in kidney (56). However, as shown in Western blot experiments, the difference in tissue glucuronidation activities is mainly due to the elevated levels of UGT2B proteins found in the liver.

The restrictive expression of UGT2B proteins in proximal tubule epithelial cells of monkey kidney cortex is not surprising because previous studies reported that UGT activity is primarily limited to the cortical region of this tissue (57), and that glucuronidation activity is greatest in cells of the proximal tubules (58, 59, 60). This specific cellular expression is consistent with the detoxifying role of UGT, because the proximal tubule cells are exposed to the initial filtrate through the glomerulus; thus it is reasonable to suggest that they play the primary role in xenobiotic conjugation (60). In addition, the expression of MR in distal tubule epithelial cells (61, 62, 63) indicates that aldosterone activity and catabolism are occurring in different cell types. The expression of the 11ß-hydroxysteroid dehydrogenase-2 enzyme in distal tubule epithelial cells (36) also suggests that, within the kidney, cortisol is inactivated into cortisone in the distal tubules and that the subsequent glucuronide conjugation is catalyzed in the proximal tubules. Such a two-cell mechanism of hormone action and metabolism has been previously described for androgen in human prostate (25). Finally, the expression of UGT2B7 in RPTEC, which is consistent with the immunohistological localization studies of UGT2B proteins, indicates that this cell type will be a helpful cellular model to further investigate the role of UGT2B7 in mineralocorticoid and glucocorticoid inactivation.

The present study identifies UGT2B7 as the major human mineralocorticoid and glucocorticoid metabolizing UGT2B enzyme. Furthermore, it also demonstrates that a single mutation in the gene encoding this enzyme affects greatly the level of aldosterone glucuronidation. Inherited disorders of aldosterone and cortisol biosynthesis are known to contribute to genetic variations in blood pressure (64), and it is reasonable to speculate that similar genetic variations in hormone metabolizing enzymes may also be involved in pathological increased serum levels of mineralocorticoids and glucocorticoids. As such, analysis of the polymorphic UGT2B7 expression in patients with apparent mineralocorticoid excess syndrome could identify UGT2B7 as a genetic factor involved in this syndrome (65).


   Acknowledgments
 
We thank Dr. Pei Min Rong, Patrick Bélanger, and Louise Berger for their technical assistance in Western blot, LC-MS/MS, and immunohistochemical analyses. Dr. Thomas R. Tephly is greatly acknowledged for the gift of the HK-293-UGT2B7 cell lines.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (to A.B.). C.G. is holder of a scholarship from the Fonds pour la formation de chercheurs et l’aide à la recherche-Fonds de la recherche en santé du Québec.

C.G. and O.B. contributed equally to this work.

Abbreviations: DHA, Dihydroaldosterone; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; HK293, human embryonic kidney 293; Km(app.), apparent Km; LC-MS/MS, liquid chromatography coupled with mass spectrometry; MR, mineralocorticoid receptor; RPTEC, renal proximal tubule epithelial cells; THA, tetrahydroaldosterone; THE, tetrahydrocortisone; UDP, uridine diphosphate; UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase; Vmax(app.), apparent maximal velocity.

Received November 18, 2002.

Accepted for publication February 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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