Human Uridine Diphosphate-Glucuronosyltransferase UGT2B7 Conjugates Mineralocorticoid and Glucocorticoid Metabolites

Abstract

Mineralocorticoid and glucocorticoid hormones are metabolized as glucuronide conjugates. Using labeled [(14)C]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 (5alpha-dihydroaldosterone and 3alpha,5beta-tetrahydroaldosterone), and both 5alpha- and 5beta-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.

Determination of mRNA Expression of Human UDP-Glucuronosyltransferases and Application for Localization in Various Human Tissues by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

“…Although GAPDH expression was slightly high in tissues, such as the testes, heart, brain, and cerebellum, almost equal copy numbers were observed in most human total RNA samples (Tables 3 and 4). Most of the UGT family enzymes were expressed in the liver and alimentary system, especially UGT2B7, an enzyme that is known to be expressed in the liver, small intestine, and colon, as well as the kidney (Parquet et al, 1985;Girard Tables 3 and 4 and Fig. 3 confirm that most of the human UGT family enzymes are expressed in the alimentary system and liver.…”

“…UGT2B4, which is known as a bile acid glucuronidating enzyme, catalyzes the glucuronide conjugation with various molecules, including bile acids, 5␣-reduced androgens, catecholestrogens (17-epiestriol and 4-hydroxyestrone), and phenolic and monoterpenoid compounds (FournelGigleux et al, 1989;Ritter et al, 1992;Pillot et al, 1993;Lévesque et al, 1999;King et al, 2000;Finel et al, 2005). Moreover, UGT2B4 is well known to be exclusively expressed in human liver and not in kidney (Pillot et al, 1993) or expressed in both liver and kidney (Girard et al, 2003). The highest expression level of UGT2B4 is quantitatively confirmed to be in the liver, whereas UGT2B4 is expressed in very low levels in heart, testis, kidney, thymus, trachea, esophagus, and placenta.…”

“…UGT2B4 and UGT2B7 are, reportedly, expressed in the kidney as well as in the liver, and UGT2B7 is responsible for glucuronidation of endogenous steroids such as gluco-and mineralocorticoids in the kidney (Girard et al, 2003). UGT2B7 was, indeed, expressed in the kidney; however, the UGT2B4 expression level in the kidney was much lower than that of UGT2B7, and the most abundantly expressed UGT in the kidney was UGT1A9, with a level 124% of UGT2B7 expression.…”

“…Numerous studies on the expression and localization of UGTs have been reported; most used reverse transcription (RT)-polymerase chain reaction (PCR) or Northern blotting (Beaulieu et al, 1996(Beaulieu et al, , 1998Lévesque et al, 1997Lévesque et al, , 1999Strassburg et al, 1997aStrassburg et al, ,b, 1998Strassburg et al, , 2000Tchernof et al, 1999;Tukey and Strassburg, 2001;Turgeon et al, 2001;Girard et al, 2003;Nakamura et al, 2008) and a few used Western blotting (Pillot et al, 1993;Ikushiro et al, 2006). In general, RT-PCR is a simple, yet sensitive, tool for detecting transcription products, whereas Western blotting can be used to verify the expression of translated proteins but requires specific antibodies for each target protein.…”

Klotho—a Common Link in Physiological and Rheumatoid Arthritis-Related Aging of Human CD4+ Lymphocytes

“…Also another postulated substrate, the steroid ␤-glucuronides, may be of interest. Although cited work described only estrogen glucuronides as experimental Klotho substrates, ␤-glucuronides are also among the main corticosteroid metabolites (54). Klotho activity, removing the glucuronide residue, was recently suggested to shift the balance toward increased levels of unconjugated, active forms of steroid hormones (10).…”