In vitro 6 beta-hydroxylation of progesterone in human renal tissue.

Increased levels of 6 beta-hydroxylated steroids have been found in the urine of several species under different physiological and pathophysiological conditions. Until now, liver, adrenal glands and placenta have been identified as organs which contain 6 beta-hydroxylase activity. However, it has not yet been established whether 6 beta-hydroxylation occurs in mammalian kidney. In this study we have performed in vitro studies with preparations from rat and human kidney cortex and have obtained evidence for the presence of a small but significant renal 6 beta-hydroxylation activity. Two points deserve to be mentioned: 1) A species difference is documented by the presence of the enzyme in human, but not in rat kidney; 2) Substrate specificity is evident. Progesterone, but not corticosterone, was transformed to the 6 beta-hydroxylated metabolite in human tissue. The biological significance of the 6 beta-hydroxylation of endogenous, as well as exogenous steroids could be that 6 beta-hydroxylation opens an alternate route of progesterone metabolism in parallel to other conversion and/or degradation pathways. Furthermore, since other 6 beta-hydroxylated steroids have been reported to exert biological effects, this may also be the case with 6 beta-hydroxy progesterone.

Interaction of 11 beta-hydroxysteroid-oxido reductase in different organs of various mammalian species.

Distribution of 11 beta-HSOR activity in different organs has been measured using tissue slices, homogenates, and microsomes. The biological material was incubated in vitro with cortisol/ cortisone (human preparations) or corticosterone/11-dehydrocorticosterone, respectively (animal preparations). Metabolites formed were quantified using RP-HPLC and on-line detection of labeled compounds. The typical pattern of CS-metabolism as obtained with rat tissue slices revealed that testis, rectum and kidney are predominant oxidizers of active gluco-CS, while liver and lung mainly function as reducers. Human placenta preparations display an exclusive oxidase activity. To trace different types of 11 beta-HSOR homogenates and microsomes of various organs were incubated with different cosubstrates (NAD+/NADH or NADP+/ NADPH, respectively). In accordance with previous reports, this study found that isoenzymes with different cosubstrate preferences exist in individual organs. 11 beta-HS oxidase activity displays a NAD+ preference in the human placenta. There was no apparent difference in cosubstrate preference between human and guinea pig placenta homogenates. In mice there is also a detectable reductive activity, whereas the Sprague-Dawley rat and golden hamster do not show any detectable activities.

Does corticosteroid metabolism in target organs affect the cardiovascular system?

The target organ metabolism of corticosteroids has been measured with biochemical and immunohistochemical techniques. Attention was focused on the key enzyme system 11 beta-hydroxysteroid oxidoreductase (11 beta-HSOR, EC 1.1.1.146). Several organs of rats, including kidney, colon, testis, pancreas, liver, lung and heart, express oxidative, as well as reductive activity, albeit with different ratios. The specific co-substrate preference in different organs points to the presence of isoforms of the enzyme, which can be inhibited by steroid compounds. In kidney tubules longitudinal heterogeneity of the enzyme distribution pattern has been reported. Inhibition of 11 beta-HSOR in transporting epithelia such as those of kidney and colon by liquorice, glycyrrhetinic acid and others leads to aldosterone-like effects of glucocorticosteroids. The mechanism of this effect is breakdown of the specific 11 beta-HSOR barrier for glucocorticosteroids which subsequently bind to mineralocorticosteroid receptors. Other possible mechanisms of interaction of the corticosteroid metabolism and the cardiovascular system are discussed.

Metabolism of dexamethasone: sites and activity in mammalian tissues.

We have used in vitro techniques to study the metabolism of dexamethasone. Tissue slices, homogenates and microsomal fractions of various mammalian organs from rats and humans have been used. We focused particularly on the question of whether or not dexamethasone (Dexa) is oxidized at the C11-OH group by 11 beta-hydroxysteroid-dehydrogenase. High activities of this enzyme system for Dexa were localized in renal cortex and rectum. Material from both human and murine liver was ineffective. The main metabolite formed from Dexa in renal and intestinal systems was identified by different mass-spectrometric techniques including on line HPLC mass spectrometry as 11-dehydro-dexamethasone. This finding was corroborated by the observation that both corticosterone and glycyrrhetinic acid block the metabolic transformation of Dexa.

Isoelectric focusing analysis of detergent extracted renal 11 beta-hydroxysteroid dehydrogenase.

11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.

Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11 beta-hydroxysteroid dehydrogenase activity.

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.

Aldosterone metabolism in rat renal tissue in vitro. Formation of lipid soluble metabolites.

In the present study the formation of lipid soluble metabolites from 3H-aldosterone was investigated in vitro in isolated kidneys and kidney and liver slices of Sprague Dawley rats. The steroids were separated by HPLC (forward and reversed phase systems) and detected on-line as UV- or 3H-chromatograms. Apart from an unenzymatically formed substance, isoaldosterone, three less polar metabolites were traced (A1, A2, A3). The structure of the quantitatively most important metabolite (A1), was identified as 5 alpha-dihydroaldosterone using a combination of techniques such as chromatographic comparison with reference steroids, antibody binding and mass spectrometry. Evidence for further conversion of DHaldo to 3 alpha, 5 alpha-tetrahydroaldosterone was obtained in chromatographic and antibody binding studies. The formation of metabolites was not dependent on glomerular filtration. Furthermore it displayed regional heterogeneity with highest activity in the outer medulla. Finally it was observed that the in vitro metabolism of aldosterone was not saturable over a range of initial aldo concentration of 10(-9) to 10(-5) M.

Corticosteroid metabolism in isolated rat kidney in vitro. I. Formation of lipid soluble metabolites from corticosterone (B) in renal tissue from male rats.

Kidneys of male Sprague Dawley rats have been isolated and perfused in vitro in order to study the metabolism of corticosterone (B). B is the main endogenous corticosteroid in this species. Using 3H-B and HPLC for the separation of steroid metabolites it has been possible to detect radioactive derivatives of B which have been denoted as met I, II and III. These substances were purified and compared with authentic reference hormones under different isocratic and gradient elution techniques. We observed chromatographic identity of met I with 11-dehydro-20-dihydro-B, of met II with 20-dihydro-B and of met III with 5 alpha-H-4,5-dihydro-B. From the fact that conversion of B can be observed with normal (50 g X l-1 albumin in perfusate) and elevated (75 g X l-1) colloid osmotic pressure of the recirculating perfusate it can be concluded that B gets access to the metabolic site in renal tissue not solely by glomerular filtration and tubular reabsorption. The metabolites identified presently are excreted in the urine. Metopirone increased the concentration of met I and decreased the concentration of met II. This is compatible with the concept of a stimulatory effect of metopirone on a C-20-hydroxysteroid oxidoreductase and a C-11-hydroxysteroid dehydrogenase.

Corticosteroid metabolism in isolated rat kidney in vitro. II. Sex dependency of metabolism and formation of 11-dehydro-corticosterone.

We have previously demonstrated that isolated kidneys from male rats convert corticosterone (B). The metabolites are formed in renal tissue, released into the recirculating perfusate and excreted in the urine (5). They have been identified as: 11-dehydro-20 zeta-dihydro-B (met I), 20 zeta-dihydro-B (met II) and 5 alpha-H-4,5-dihydro-B (met III), using HPLC. In view of sex dependency of hepatic corticosteroid metabolism we have presently investigated whether or not equivalent sexual differences exist in renal tissue. In applying appropriate HPLC-techniques we could demonstrate a fourth metabolite formed from B, which was chromatographically identical with 11-dehydro-B (= met IV). Female rat kidneys form predominantly the less polar metabolites III and IV, in contrast to kidneys from male rats, which produce met III and the more polar metabolites I and II.

Corticosteroid metabolism in isolated rat kidney in vitro. III. Structure analysis of lipid soluble metabolites of corticosterone.

We have previously demonstrated that isolated rat kidneys in vitro convert corticosterone (B). Four lipid soluble metabolites (met I, II, III and IV) have been identified which differ in polarity from the parent hormone [2, 5, 6, 11, 15, 16]. In the present experiments these metabolites have been extracted from perfusate after 4 h of recirculation through isolated kidneys of male and female rats. Subsequently they have been separated by HPLC using a polar stationary phase system and n-hexane and isopropanol as eluents. The chromatographic comparison of met II with authentic 20 alpha- and 20 beta-isomers documented that met II is identical with 20 beta-dihydro-B. Measurements of the mass spectra of the purified samples revealed the following structures: met I = 20 beta-dihydro-11-dehydro-B, met II = 20 beta-dihydro-B, met III = 5 alpha-H-4,5-dihydro-B and met IV = 11-dehydro-B.

Renal metabolism of corticosteroid hormones.

IK and STF from male and female rats have been used to study in vitro the renal metabolism of B. in male rat tissue four lipid soluble metabolites (I-IV) have been found, I + II being more polar and III + IV being less polar than B. I and II have been identified as 11-dehydro-20-hydroxy-B and 20-hydroxy-B. The structure of III and IV remains to be determined. Renal tissue from female rats produced predominantly III indicating sexual variations of steroid metabolism in kidneys.--The literature has been reviewed which documents that the kidneys in addition to B metabolize A, cortisol, progesterone and other corticosteroids.