Abstract

In the human and in rodents like the rat and mouse, the liver enzyme 11β-hydroxysteroid dehydrogenase type I (11β-HSD-I) is a functional oxidoreductase preferring NADP⁺/NADPH as cosubstrate, while the renal isoenzyme (11β-HSD-II) prefers NAD⁺ as cosubstrate, and seems to be a pure oxidase and protects the tubular mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11β-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10⁻⁶ mol/l cortisol, liver and kidney microsomes preferred NAD⁺ to NADP⁺ (10⁻³ mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with K _m values of 0·64 μmol/l and 9·8 μmol/l with NAD⁺ and NADP⁺ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate K _m was 7·2 μmol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar K _m and V_max values for NAD⁺ to NADP⁺ as cosubstrates (K _m of 4·3 μmol/l and 5·0 μmol/l respectively). The NAD⁺ preference for the oxidation of 10⁻⁶ mol/l cortisol described above may be due to a second, NAD⁺-preferring 11β-HSD with a K _m of 1·4 μmol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (K _m: 1·2 μmol/l; V_max: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11β-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD⁺-preferring. This kidney enzyme (analogous to 11β-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig.

Journal of Endocrinology (1997) 153, 291–298

Due to high binding affinity of progesterone to the human mineralocorticoid receptor (hMR), progesterone competes with the natural ligand aldosterone. In order to analyse how homeostasis can be maintained by mineralocorticoid function of aldosterone at the MR, especially in the presence of elevated progesterone concentrations during the luteal phase and pregnancy, we investigated protective mechanisms such as the decrease of free progesterone by additional binding sites and progesterone metabolism in renal cells. As a prerequisite for sequestration of progesterone by binding to the human progesterone receptor (hPR) we demonstrated the existence of hPR expression in female and male kidney cortex and medulla at the level of transcription and translation. We identified hPR RNA by sequencing the RT-PCR product and characterised the receptor by ligand binding and scatchard plot analysis. The localisation of renal hPR was shown predominantly in individual epithelial cells of distal tubules by immunohistology, and the isoform hPR-B was detected by Western blot analysis. As a precondition for renal progesterone metabolism, we investigated the expression of steroid-metabolising enzymes for conversion of progesterone to metabolites with lower affinity to the hMR. We identified the enzyme 17alpha-hydroxylase for renal 17alpha-hydroxylation of progesterone. For 20alpha-reduction, different hydroxysteroid dehydrogenases (HSDs) such as 20alpha-HSD, 17beta-HSD type 5 (3alpha-HSD type 2) and 3alpha-HSD type 3 were found. Further, we detected the expression of 3beta-HSD type 2 for 3beta-reduction, 5alpha-reductase (Red) type 1 for 5alpha-reduction, and 5beta-Red for 5beta-reduction of progesterone in the human kidney. Therefore metabolism of progesterone and/or binding to hPR could reduce competition with aldosterone at the MR and enable the mineralocorticoid function.

The 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) convert cortisol to its inactive metabolite cortisone and vice versa. 11beta-HSD type 1 (11beta-HSD-1) functions as a reductase in vivo, regulating intracellular cortisol levels and its access to the glucocorticoid receptor. In contrast, 11beta-HSD-2 only mediates oxidation of natural glucocorticoids, and protects the mineralocorticoid receptor from high cortisol concentrations. We investigated the in vivo and in vitro effects of ACTH on the recently characterized 11beta-HSDs in guinea pig liver and kidney. Tissue slices of untreated guinea pigs were incubated with (3)H-labelled cortisol or cortisone and ACTH(1-24) (10(-10) and 10(-9) mol/l). The 11beta-HSD activities in liver and kidney slices were not influenced by in vitro incubation with ACTH(1-24). In addition, guinea pigs were treated with ACTH(1-24) or saline injections s.c. for 3 days. Liver and kidney tissue slices of these animals were incubated with (3)H-labelled cortisol or cortisone. In vivo ACTH treatment significantly increased reductase and decreased oxidase activity in liver and kidney. Furthermore, 11beta-HSD-1 activity assessed by measurement of the urinary ratio of (tetrahydrocortisol (THF)+5alphaTHF)/(tetrahydrocortisone) was significantly increased after ACTH treatment compared with the control group. Plasma levels of cortisol, cortisone, progesterone, 17-hydroxyprogesterone and androstenedione increased significantly following in vivo ACTH treatment. The enhanced reductase activity of the hepatic and renal 11beta-HSD-1 is apparently caused by cortisol or other ACTH-dependent steroids rather than by ACTH itself. This may be an important fine regulation of the glucocorticoid tonus for stress adaptation in every organ, e.g. enhanced gluconeogenesis in liver.