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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2803-2809
Copyright © 2003 by The Endocrine Society

The Human Kidney Is a Progesterone-Metabolizing and Androgen-Producing Organ

M. Quinkler, C. Bumke-Vogt, B. Meyer, V. Bähr, W. Oelkers and S. Diederich

Department of Endocrinology, Klinikum Benjamin Franklin, Free University, 12200 Berlin, Germany

Address all correspondence and requests for reprints to: Dr. Marcus Quinkler, Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH. E-mail: m.o.quinkler{at}bham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone (P) is a potent antagonist of the human mineralocorticoid receptor (MR) in vitro. We have previously demonstrated effective downstream metabolism of P in the kidney. This mechanism potentially protects the MR from P action. Here, we have investigated the expression and functional activity of steroidogenic enzymes in human kidney. RT-PCR analysis demonstrated the expression of 5{alpha}-reductase type 1, 5ß-reductase, aldo-keto-reductase (AKR) 1C1, AKR1C2, AKR1C3, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) type 2, and 17{alpha}-hydroxylase/17,20-lyase (P450c17). The presence of 3ß-HSD type 2 and P450c17 indicated that conversion of pregnenolone to dehydroepiandrosterone (DHEA) and to androstenedione may take place effectively in kidney. To investigate this further, we incubated kidney subcellular fractions with radiolabeled pregnenolone. This resulted in efficient formation of DHEA from pregnenolone, indicating both 17{alpha}-hydroxylase and 17,20-lyase activities exerted by P450c17. Radiolabeled DHEA was converted via androstenedione, androstenediol, and testosterone, indicating both 3ß-HSD type 2 activity and 17ß-HSD activity. In addition, the conversion of testosterone to 5{alpha}-dihydrotestosterone was detectable, indicating 5{alpha}-reductase activity. In conclusion, we verified the expression and functional activity of several enzymes involved in downstream metabolism of P and androgen synthesis in human kidney. These findings may be critical to the understanding of water balance during the menstrual cycle and pregnancy and of sex differences in hypertension.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE (P) IS a strong mineralocorticoid receptor (MR) antagonist in vitro due to its high binding affinity to the MR (1, 2, 3, 4). However, there does not appear to be a significant anti-MR effect in vivo even with high P concentrations, e.g. luteal phase (30–110 nmol/liter) and pregnancy (320–700 nmol/liter) (5, 6). Until recently, it was unknown how aldosterone maintained its function as an effective MR agonist in the presence of a 100-fold excess of P and why there were not more anti-MR effects, e.g. enhanced diuresis, lowering of blood pressure, and electrolyte disturbances. Previous explanations were not satisfactory, e.g. higher plasma protein binding of P or higher instability of the P-MR complex. We have recently shown that the kidney is able to effectively metabolize P to downstream metabolites, such as 17{alpha}-hydroxyprogesterone (17{alpha}-OH-P), 20{alpha}-dihydroprogesterone (20{alpha}-DH-P), and ring A reduced metabolites (7, 8), which possess weaker inhibitory activity than P at the MR (4). This may be the mechanism responsible for protection of the MR from P action, similar to the inactivating metabolism of cortisol to cortisone by 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 2 (9, 10, 11).

This study investigated which enzymes are responsible for downstream conversion of P in the human kidney. Furthermore, the enzymes identified suggested that these enzymes may also be involved in the conversion of other steroid substrates, e.g. precursors of androgen synthesis, in human kidney. Therefore, we explored the functional activity of steroidogenic enzymes expressed in human kidney and potentially involved in androgen synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Radiolabeled and unlabeled steroids

[7-3H]Pregnenolone ([7-3H]Preg; 10–25 Ci/mmol), [4-14C]P (0.02 mCi/ml), and [4-14C]dehydroepiandrosterone ([4-14C]DHEA; 0.02 mCi/ml) were purchased from NEN Life Science Products (Boston, MA).

Unlabeled P (4-pregnen-3,20-dione), deoxycorticosterone, 20{alpha}-DH-P (4-pregnen-20{alpha}-ol-3-one), 5{alpha}-DH-P (5{alpha}-pregnan-3,20-dione), 3ß,5{alpha}-TH-P (5{alpha}-pregnan-3ß-ol-20-one), 20{alpha}-DH,5{alpha}-DH-P (5{alpha}-pregnan-20{alpha}-ol-3-one), 16{alpha}-OH-P (4-pregnen-16{alpha}-ol-3,20-dione), 5ß-DH-P (5ß-pregnan-3,20-dione), 20{alpha}-DH,3ß,5{alpha}-TH-P (5{alpha}-pregnan-3ß,20{alpha}-diol), 20{alpha}-DH,3{alpha},5{alpha}-TH-P (5{alpha}-pregnan-3{alpha},20{alpha}-diol), 3{alpha},5{alpha}-TH-P (5{alpha}-pregnan-3{alpha}-ol-20-one), 11{alpha}-OH-P (4-pregnen-11{alpha}-ol-3,20-dione), 11ß-OH-P (4-pregnen-11ß-ol-3,20-dione), 20{alpha}-DH,3{alpha},5ß-TH-P (5ß-pregnan-3{alpha},20{alpha}-diol), androstenedione (4-dione; 4-androsten-3,17-dione), DHEA (5-androsten-3ß-ol-17-one), Preg (5-pregnen-3ß-ol-20-one), 17{alpha}-hydroxypregnenolone (17{alpha}-OH-Preg; 3ß,17{alpha}-dihydroxy-5-pregnen-20-one), testosterone (T; 4-androsten-17ß-ol-3-one), estrone (1,3,5(10)-estratrien-3-ol-17-one), 17ß-estradiol (1,3,5(10)-estratrien-3{alpha},17ß-diol), 5{alpha}-dihydrotestosterone (5{alpha}-DH-T; 5{alpha}-androstane-17ß-diol-3-one), 5ß-DH-T (5ß-androstane-17ß-diol-3-one), 3{alpha}-androstanediol (3{alpha}-diol; 5{alpha}-androstane-3{alpha},17ß-diol), androstanedione (5{alpha}-androstane-3,17-dione), androstenediol (5-androstene-3ß,17ß-diol), and androsterone (5{alpha}-androstane-3{alpha}-ol,17ß-one) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). 17{alpha}-OH-P (4-pregnen-17{alpha}-ol-3-one) was purchased from Makor Chemicals Ltd. (Jerusalem, Israel). 17{alpha}-OH,20{alpha}-DH-P (4-pregnen-17,20{alpha}-diol-3-one) and 3{alpha},5ß-TH-P (5ß-pregnan-3{alpha}-ol-20-one) were obtained from Paesel \|[amp ]\| Lorei (Hanau, Germany), and 6ß-OH-P (4-pregnen-6ß-ol-3,20-diol) was obtained from Steraloids, Inc. (Wilton, NH).

Preparation of subcellular fractions from human kidney tissue

Human kidney specimens from tumor nephrectomies of postmenopausal women were obtained and prepared as described previously (7, 8). Ethical approval was granted by the local ethic committee. Renal cortex and medulla were divided macroscopically, cut into small pieces, and homogenized for preparation of subcellular fractions or for RNA preparation. The preparation of subcellular fractions was performed as described by Lakhsmi et al. (12). Total protein content was estimated by the Bradford method (Bio-Rad Laboratories, Inc., Munich, Germany).

Incubations with kidney subcellular fractions

We performed preliminary studies for time kinetics, protein kinetics, and cosubstrate preferences (data not shown) to determine optimum conditions for incubation of subcellular fractions. Both cytosolic and microsomal incubations were carried out in 1 ml 0.01 M sodium phosphate buffer (pH 7.0 for reduction and pH 8.1 for oxidation) and, in addition, a coenzyme-regenerating system (10-2 M glucose-6-phosphate, 10 U glucose-6-phosphate-dehydrogenase; Roche, Mannheim, Germany). All incubations were carried out at 37 C in a shaking water bath; all experiments were performed in triplicate.

Incubation assay for the cytosolic fraction. We incubated 4 mg cytosolic protein from kidney cortex and medulla with 10-3 mol/liter NADPH and 200,000 cpm [4-14C]P and [4-14C]DHEA, respectively, for 120 min. We investigated the following unlabeled steroids as substrates (10-4 M) under the same conditions using NADPH as a cosubstrate for reduction, NADP+ for oxidation of 3{alpha},5ß-TH-P, 20{alpha}-DH,3{alpha},5ß-TH-P, 20{alpha}-DH,5ß-DH-P, 20{alpha}-DH-P, T, 5{alpha}-DH-T, 3{alpha}-diol, 4-dione, and estrone. For oxidation of 3{alpha}-diol, NAD+, rather than NADP+, was added. Enzyme kinetic analysis for 20{alpha}-reduction of P was performed using increasing concentrations of unlabeled P (10-8–10-5 M) in addition to radiolabeled P within the linear time range of the enzymatic reaction (40 min of incubation).

Incubation assay for the microsomal fraction. One milligram of microsomal protein isolated from kidney cortex and medulla was incubated with 10-3 M NADPH (for reduction, pH 7.0) or NAD+/NADP+ (for oxidation, pH 8.1); 200,000 cpm [4-14C]P, [7-3H]Preg, or [4-14C]DHEA were added, and incubations were carried out for 60, 120, and 120 min, respectively, thus ensuring that incubations were within the linear time frame of the enzymatic reactions. In addition, 10-4 M 4-dione, T, Preg, and DHEA were incubated with the microsomal fraction under the same conditions for 120 min with NADPH as cosubstrate

Steroid extraction and detection of steroids by thin layer chromatography (TLC)

The incubations were stopped, and steroids were extracted from the incubations with methylacetate and separated by two-dimensional TLC (first dimension, 50 min in 35/65 methylacetate/ethylendichloride; second dimension with 25:75 hexanol/hexane for 210 min) as described previously (7, 8) and shown in Fig. 1Go. For detection of T, 5{alpha}-DH-T, and 5ß-DH-T from microsomal incubations, we used one-dimensional TLC with 25:75 hexanol/hexane for 210 min.



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  		Figure 1. Separation of steroids with two-dimensional TLC. The first dimension was run with 35:65 methylacetate/ethylendichloride for 100 min; the second dimension run was performed with 25:75 hexanol/hexane for 210 min. This figure shows the incubation of microsomes from postmenopausal renal cortex with [3H]Preg. A, TLC plate after staining with Liebermann-Burchard reagent and heating, revealing the unlabeled control steroids (no. 1–7). B, 3H scan of the same TLC plate. 1) Preg; 2) DHEA; 3) 17{alpha}-OH-Preg; 4) P; 5) 4-dione; 6) 17{alpha}-OH-P; 7) T; 8 and 9) unknown substances. S, Starting point.

 
RNA preparation and RT-PCR

Total RNA was prepared from four human postmenopausal kidneys using the RNeasy MIDI kit (Amersham Pharmacia Biotech, Freiburg, Germany). Single-stranded cDNA was synthesized by Superscript II-RT (Life Technologies, Inc., Karlsruhe, Germany) and random hexamer primers, using 2.5 µg total RNA as a template. cDNA (40 ng) was then subjected to PCR amplification with 12 pmol/liter specific primers for the steroidogenic enzymes of interest (TIB MOLBIOL, Berlin, Germany; GENSET SA, Paris, France) overspanning at least one intron (Table 1Go) in a volume of 25 µl Tris-buffer with 1 U Taq polymerase. PCR conditions were 35 cycles for 45 sec at 94 C, for 1 min at 55–65 C (dependent on the melting temperature of the respective primer pairs), and for 1.5 min at 72 C. Each RT-PCR analysis was performed using an additional negative control and two positive controls, human adrenal cortex and human testis (each 40 ng cDNA; Fig. 2Go). RT-PCR products were gel-purified and subjected to direct sequencing for verification.


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  		Table 1. Gene-specific forward and reverse primers, the location of the primers on their genes, and the resulting amplified DNAs

 


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  		Figure 2. RT-PCR analysis of the expression of different enzymes in postmenopausal kidney (ki), human adrenal (adr), and human testis (test). lad, Control protein ladder; neg, negative control; cyt b5, cytochrome b5; 5{alpha}-red-1, 5{alpha}-reductase type 1; 5{alpha}-red-2, 5{alpha}-reductase type 2; 5ß-red, 5ß-reductase.

 
For specific identification of 17ß-HSD type 5 [aldo-keto-reductase (AKR) 1C3], we used three forward and reverse primers spanning across all nine exons of the gene (Table 1Go). The sequence of 17ß-HSD type 5 (AKR1C3) differs only in amino acids 75 and 175 from the sequence of 3{alpha}-HSD type 2 (13, 14). These two different amino acids at positions 75 and 175 do not change the enzymatic properties, and it is suggested that these are identical enzymes (13, 15, 16).

Statistics

For the calculation of the Michaelis-Menten constant (Km) and the maximal reaction velocity (Vmax), we used the Eadie-Hofstee transformation. In addition, the intrinsic clearance value (CLint = Vmax/Km) was calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Conversion of P and its metabolites

To characterize the enzymes involved in downstream conversion of P, we incubated subcellular fractions of human kidney with labeled P or unlabeled P metabolites. In the cytosolic fraction we found more than 80% conversion of P to 20{alpha}-DH-P, the only metabolite found in cytosol, after 4 h of incubation. The Km for this reaction was 11.2 µmol/liter, the Vmax was 19.4 nmol/min·mg, and the intrinsic clearance value (Vmax/Km) was 1.63 ml/min·mg. Consistent with the observed efficient conversion of P to 20{alpha}-DH-P, we detected 20-reduction of 3{alpha},5ß-TH-P to 20{alpha}-DH,3{alpha},5ß-TH-P, but we could not demonstrate 20-oxidation of various other P metabolites (Table 2Go). The presence of enzymes with major 20{alpha}-reductase activity, 20{alpha}-HSD (AKR1C1) and 17ß-HSD type 5 (AKR1C3), was demonstrated by RT-PCR (Fig. 2Go).


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  		Table 2. Summary of detected conversion of different unlabeled steroids in cytosolic and microsomal fraction of human postmenopausal kidneys

 
After incubation of the microsomal fraction from human kidney with radiolabeled P, we detected 5{alpha}-DH-P, 20{alpha}-DH,5{alpha}-DH-P, and 3ß,5{alpha}-TH-P, indicating 5{alpha}-reductase activity. The expression of 5{alpha}-reductase type 1 was demonstrated by RT-PCR, whereas 5{alpha}-reductase type 2 was not detected (Fig. 2Go). Generation of 3ß,5{alpha}-TH-P from P suggests that the kidney also expresses 3ß-HSD activity. Consistent with this finding we detected 3ß-HSD type 2 mRNA. However, there was no evidence of 3ß-HSD type 1 expression (Fig. 2Go). In contrast to what was shown previously for premenopausal kidney (8), there was no conversion to 3{alpha}- or 5ß-reduced P metabolites in the postmenopausal kidney tissue used for the current experiments. Despite this lack of functional activity, we still were able to detect the expression of 3{alpha}-HSD type 3 (AKR1C2) mRNA and 5ß-reductase mRNA in postmenopausal kidney samples (Fig. 2Go).

In the microsomal fraction we found predominant conversion of P to 17{alpha}-OH-P and 17{alpha}-OH,20{alpha}-DH-P, but also to 16{alpha}-OH-P. This suggested the presence of 17{alpha}-hydroxylase/17,20-lyase (P450c17), which could be demonstrated by RT-PCR (Fig. 2Go). Sequencing of the RT-PCR products (exons 1–3) revealed 100% identity to the published P450c17 sequence.

Conversion of Preg and DHEA

The expression and functional activity of P450c17, 3ß-HSD type 2, and 5{alpha}-reductase type 1 suggested that not only may these enzymes contribute to progesterone metabolism, but that efficient generation of androgens may occur in human kidney. To investigate this in further detail, we incubated both cytosolic and microsomal fractions with radiolabeled Preg or DHEA and in addition with unlabeled T, estrone, 4-dione, 5{alpha}-DH-T, or 3{alpha}-diol.

In microsomes we detected the conversion of [3H]Preg to 17{alpha}-OH-Preg (mean ± SD, 21.6 ± 2.1%) and DHEA (18.0 ± 1.5%), indicating the presence of both 17{alpha}-hydroxylase and 17,20-lyase activities of P450c17 (Figs. 1Go and 3aGo). One metabolite peak on the TLC plate could not be identified (mean ± SD, 10.8 ± 1.2% conversion; Fig. 1Go). In addition, we detected traces of androstenediol in the microsomal fraction, reflecting downstream conversion of DHEA via 17ß-HSD activity (Fig. 3aGo). The 17,20-lyase activity of P450c17, which catalyzes the conversion of 17{alpha}-OH-Preg to DHEA, requires allosteric facilitation of the interaction of P450c17 with its electron donor P450 oxidoreductase. This allosteric modulation is mediated by cytochrome b5 (17, 18), and cytochrome b5 expression was readily detected by RT-PCR in both human kidney cortex and medulla (Fig. 2Go).



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  		Figure 3. Schematic representation of sex steroid formation and the involved enzymes in human postmenopausal kidney microsomes (A) and cytosol (B). The thickness of the arrows represents the approximate quantity of conversion. Red, Reductase.

 
Microsomal incubations with radiolabeled DHEA and NADPH or NADH as cosubstrate revealed minimal conversion of DHEA to 4-dione (0.5–1%) and androstenediol (0.8–1%), indicating 3ß-HSD and 17ß-HSD activities (Fig. 3aGo).

After 120-min incubation of kidney microsomes with 10-4 M unlabeled T, we did not detect 5{alpha}- or 5ß-DH-T. However, there was conversion of 4-dione to T and in small amounts also to androstanedione (Table 2Go and Fig. 3aGo).

In the cytosolic kidney fraction, radiolabeled DHEA was predominantly converted to androstenediol (mean ± SD, 46.9 ± 0.9% conversion), and to two other unknown metabolites (1–3% conversion). These unknown metabolites were not identical to the steroids tested (see Materials and Methods). The conversion to androstenediol indicates a strong 17ß-HSD activity. In accordance with this, we found efficient conversion of 4-dione to T in human kidney cytosol (Table 2Go and Fig. 3bGo). 17ß-HSD activity could be due to all three enzymes of the AKR family (AKR1C1, AKR1C2, and AKR1C3), for which expression was detected in the kidney and which all exhibit 17ß-HSD activity. However, 17ß-HSD type 5 (AKR1C3) is the most probable enzyme due to its high 17ß-HSD activity and widespread cytosolic distribution.

Cytosolic incubations also revealed a small conversion of T to 5{alpha}-DH-T, indicating 5{alpha}-reductase activity by 5{alpha}-reductase type 1 (Table 2Go and Fig. 2Go). 5{alpha}-DH-T was inactivated to 3{alpha}-diol (Fig. 3bGo) by 3{alpha}-HSD activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has long been known that P has potential to interfere with signaling through the MR. This may be relevant to the physiological and pathophysiological changes in fluid balance and blood pressure seen in pregnancy and possibly in the luteal phase of the menstrual cycle or in women taking contraceptives containing synthetic progestins or P itself. P binds in vitro with similar affinity to the MR as aldosterone, but confers only weak agonistic MR activity (1, 2, 3, 4). This anti-MR potency of P results in increased urinary sodium excretion if given im in large amounts (19, 20, 21, 22). During pregnancy, P concentrations exceed those of aldosterone at least 50- to 100-fold. It was not known how aldosterone, with a slight increase in plasma concentration during pregnancy, can act as an MR agonist in situations with high P concentrations. However, P has 10-fold higher plasma protein binding than aldosterone, thereby reducing the unbound steroid fraction of P (23). In addition, P dissociates faster from the MR complex than aldosterone due to a higher stability of the aldosterone-MR complex (2). We believe that a potent and effective P metabolism to inactive metabolites (4) in human kidney could be an equivalent protective mechanism for the protection of the MR similar to the inactivation of cortisol by 11ß-HSD type 2 (7, 8). In a previous study we demonstrated that even at high P concentrations the kidney efficiently converted P to downstream metabolites (7). In this study we identified the enzymes responsible for this P metabolism. This is of special interest, because an alteration of P metabolism could be one cause of hypertension and water retention in pregnancy and preeclampsia (7). Here, we used kidney samples from postmenopausal women. However, it should be taken into account that the activation and inactivation of steroid hormones may vary with the sex and age of the donor.

Strong 20{alpha}-reduction of P may be due to the catalytic activity of AKR1C1 and/or AKR1C3, which were both expressed in human kidney. It appears that the AKR1C1 (20{alpha}-HSD) is the most probable enzyme to catalyze 20{alpha}-reduction (24). We calculated a Km for 20{alpha}-reduction of P similar to reports in AKR1C1-transfected cells (2.6–18 µmol/liter) (25, 26). However, AKR1C3 (3{alpha}-HSD type 2 = 17ß-HSD type 5) may also contribute to the conversion of P to 20{alpha}-DH-P, because it exerts strong 20{alpha}-HSD activity in addition to its 17ß-HSD activity (13, 24, 26, 27, 28).

We also found efficient 17{alpha}-hydroxylation of P in human kidney. The microsomal P450c17 catalyzes 17{alpha}-hydroxylation, followed by 17,20-lysis of 17{alpha}-hydroxylated products (29), and is expressed in human steroidogenic tissues. Our results are another example of P450c17 expression in extraadrenal and extragonadal tissues. Besides 17{alpha}- and 16{alpha}-hydroxylation of P (30, 31, 32), we detected the expression of both P450c17 and cytochrome b5 in human adult kidney, resulting not only in efficient 17{alpha}-hydroxylase activity, but also in 17,20-lyase activity of P450c17. Accordingly, we found the conversion of Preg via 17{alpha}-OH-Preg to DHEA. Thus, the kidney seems to be the only tissue, except for adrenal, gonad, placenta, and brain, that expresses functionally active P450c17, and the physiological relevance of this finding needs further investigation.

Further downstream, the conversion of DHEA to 4-dione is catalyzed by 3ß-HSD. In human kidney we found the expression of 3ß-HSD type 2 converting 5{alpha}-DH-P to 3ß,5{alpha}-TH-P. However, we did not detect the conversion of Preg to P and found only weak conversion of DHEA to 4-dione in human kidney. Therefore, 3ß-HSD type 2 seems to play a role in renal steroid metabolism rather than steroidogenesis.

We detected a strong conversion of 4-dione to T (17ß-HSD activity) in renal cytosol and therefore assume that this reaction is specific for AKR1C3 (17ß-HSD type 5) because we could not detect 17ß-HSD type 3 mRNA in human kidney. We also demonstrated 5{alpha}-reductase type 1 expression in human kidney tissue, which was snap-frozen immediately. In contrast to our finding, Thigpen et al. (33) were not able not detect 5{alpha}-reductase type 1 mRNA; however, they used postmortem kidneys. We found functional activity of 5{alpha}-reductase type 1 resulting in 5{alpha}-reduction of P to 5{alpha}-DH-P, of 4-dione to androstanedione, and of T to the potent androgen 5{alpha}-DH-T. Further studies are necessary to investigate the local production of 5{alpha}-DH-T within the kidney, its possible influence on water and salt retention, and its impact on the pathogenesis of hypertension. In addition, 5{alpha}-metabolites of P exert various biological functions, such as anxiolytic properties, relaxing potency of smooth muscle cells, and neuroactive {gamma}-aminobutyric acid A receptor agonistic properties, e.g. 3{alpha},5{alpha}-TH-P (34, 35, 36, 37). Thus, 5{alpha}-reduction could also represent an important modulatory activity with regard to P metabolism.

We suggest that the described P metabolism in MR target cells is a possible protection mechanism for the MR. Therefore, these enzymes may be relevant to physiological and pathophysiological changes in fluid balance and blood pressure. An alteration of expression of these enzymes may play a role in pregnancy-induced hypertension, preeclampsia, and the premenstrual syndrome.

In vitro, P has a positive effect on the relaxation of smooth muscle cells within the uterus as well as in extrauterine tissue (38). Therefore, it may be possible that P regulates renal blood flow, reducing the renal effect of angiotensin II and increasing the glomerular filtration rate (39). The underlying mechanism is still unknown, but P-metabolizing enzymes may play a regulatory role in renal blood flow.

In addition, it was recently shown that P enhances calcium reabsorption in distal tubules and collecting duct of rabbit kidney (40), probably mediated by the P receptor, which is expressed in the kidney (40). Renal P-metabolizing enzymes may regulate the access of P to the P receptor and consequently alter the effect of P on calcium homeostasis.

In postmenopausal women all sex steroids are of extragonadal origin. Several tissues produce androgens and estrogens from precursor steroids secreted by the adrenals (e.g. DHEA and DHEA sulfate). We investigated the synthesis and metabolism of androgenic steroids in human postmenopausal kidneys and were able to characterize in detail a complex steroid enzymology in the human kidney. The kidney is able to synthesize DHEA from Preg via 17{alpha}-OH-Preg, with further downstream activation via 4-dione to T and 5{alpha}-DH-T. The physiological importance of androgen generation in the kidney remains to be elucidated. Androgens appear to play an important role in the pathogenesis of hypertension and in modulating sex-specific differences in hypertension. This hypothesis is supported by the finding that castration of spontaneously hypertensive male rats or treatment with the androgen receptor antagonist flutamide lowers blood pressure and slows the progression of hypertensive organ damage to the kidney (41). Administration of T to these castrated rats or to female rats resulted in increases in blood pressure to the level in untreated male rats, and renal injury subsequently showed fast progression. This effect could be mediated directly via activation of the renin-angiotensin system or via increased proximal tubular reabsorption (41, 42, 43).

In conclusion, we clearly established the effective generation of androgens in the human kidney by locally expressed steroidogenic enzymes, and it remains an important goal of future research to understand the physiological role of androgen formation in the kidney.


   Acknowledgments
 
We thank K. Miller (Departments of Urology and Pathology, Klinikum Benjamin Franklin, Free University, Berlin, Germany) for the human kidney tissues. We are indebted to S.-M. Herrmann (Department of Clinical Pharmacology, Klinikum Benjamin Franklin, Free University, Berlin, Germany) for sequence analysis of PCR products. We thank J. Lepenies (Berlin, Germany), M. S. Cooper (Birmingham, UK), and W. Arlt (Birmingham, UK) for help with the manuscript. We are grateful to W. Arlt for provision of cDNA from human adrenal and testis.


   Footnotes
 
This work was supported by Research Grant DI 741/1-3 from the Deutsche Forschungsgemeinschaft (to S.D.) and Deutsche Forschungsgemeinschaft Postdoctoral Research Fellowship Grant QU142/1-1 (to M.Q.).

Abbreviations: AKR, Aldo-keto-reductase; 20{alpha}-DH-P, 20{alpha}-dihydroprogesterone; DHEA, dehydroepiandrosterone; DH-T, dihydrotestosterone; 3{alpha}-diol, 3{alpha}-androstanediol; 4-dione, androstenedione; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; 17{alpha}-OH-P, 17{alpha}-hydroxyprogesterone; 17{alpha}-OH-Preg, 17{alpha}-hydroxypregnenolone; P, progesterone; P450c17, 17{alpha}-hydroxylase/17,20-lyase; Preg, pregnenolone; T, testosterone; 3ß,5{alpha}-TH-P, 5{alpha}-pregnan-3ß-ol-20-one; TLC, thin layer chromatography; Vmax, maximal reaction velocity.

Received December 13, 2002.

Accepted March 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rupprecht R, Reul JM, van Steensel B, Soder M, Berning B, Holsboer F, Damm K 1993 Pharmacological and functional characterization of human mineralocorticoid and glucocorticoid receptor ligands. Eur J Pharmacol 247:145–154[CrossRef][Medline]
  2. Souque A, Fagart J, Couette B, Davioud E, Sobrio F, Marquet A, Rafestin Oblin ME 1995 The mineralocorticoid activity of progesterone derivatives depends on the nature of the C18 substituent. Endocrinology 136:5651–5658[Abstract]
  3. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268–275[Abstract/Free Full Text]
  4. Quinkler M, Meyer B, Bumke-Vogt C, Grossmann C, Gruber U, Oelkers W, Diederich S, Bähr V 2002 Agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor. Eur J Endocrinol 146:789–799[Abstract]
  5. Johansson ED, Jonasson LE 1971 Progesterone levels in amniotic fluid and plasma from women. I. Levels during normal pregnancy. Acta Obstet Gynecol Scand 50:339–343[Medline]
  6. Rosenthal HE, Slaunwhite WR, Sandberg AA 1969 Transcortin: a corticosteroid-binding protein of plasma. X. Cortisol and progesterone interplay and unbound levels of these steroids in pregnancy. J Clin Endocrinol Metab 29:352–367[Abstract/Free Full Text]
  7. Quinkler M, Johanssen S, Grossmann C, Bähr V, Müller M, Oelkers W, Diederich S 1999 Progesterone metabolism in the human kidney and inhibition of 11ß-hydroxysteroid dehydrogenase type 2 by progesterone and its metabolites. J Clin Endocrinol Metab 84:4165–4171[Abstract/Free Full Text]
  8. Quinkler M, Johanssen S, Bumke-Vogt C, Oelkers W, Bähr V, Diederich S 2001 Enzyme-mediated protection of the mineralocorticoid receptor against progesterone in the human kidney. Mol Cell Endocrinol 171:21–24[CrossRef][Medline]
  9. Funder JW, Pearce PT, Smith R, Smith AI 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242:583–585[Abstract/Free Full Text]
  10. Edwards CR, Stewart PM, Burt D, Brett L, McIntyre MA, Sutanto WS, de Kloet ER, Monder C 1988 Localisation of 11ß-hydroxysteroid dehydrogenase–tissue specific protector of the mineralocorticoid receptor. Lancet 2:986–989[CrossRef][Medline]
  11. Quinkler M, Oelkers W, Diederich S 2001 Clinical implications of glucocorticoid metabolism by 11ß-hydroxysteroid dehydrogenases in target tissues. Eur J Endocrinol 144:87–97[Abstract]
  12. Lakshmi V, Monder C 1988 Purification and characterization of the corticosteroid 11ß-dehydrogenase component of the rat liver 11ß-hydroxysteroid dehydrogenase complex. Endocrinology 123:2390–2398[Abstract/Free Full Text]
  13. Dufort I, Rheault P, Huang X-F, Soucy P, Luu-The V 1999 Chararcteristics of a highly labile human type 5 17ß-hydroxysteroid dehydrogenase. Endocrinology 140:568–574[Abstract/Free Full Text]
  14. Lin HK, Jez JM, Schlegel BP, Peehl DM, Pachter JA, Penning TM 1997 Expression and characterization of recombinant type 2 3{alpha}-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3{alpha}/17ß-HSD activity and cellular distribution. Mol Endocrinol 11:1971–1984[Abstract/Free Full Text]
  15. Dufort I, Labrie F, Luu-The V 2001 Human types 1 and 3 3{alpha}-hydroxysteroid dehydrogenases: differential lability and tissue distribution. J Clin Endocrinol Metab 86:841–846[Abstract/Free Full Text]
  16. Penning TM, Burczynski ME, Jez JM, Lin HK, Ma H, Moore M, Ratnam K, Palackal N 2001 Structure-function aspects and inhibitor design of type 5 17ß-hydroxysteroid dehydrogenase (AKR1C3). Mol Cell Endocrinol 171:137–149[CrossRef][Medline]
  17. Katagiri M, Kagawa N, Waterman MR 1995 The role of cytochrome b5 in the biosynthesis of androgens by human P450c17. Arch Biochem Biophys 317:343–347[CrossRef][Medline]
  18. Auchus RJ, Lee TC, Miller WL 1998 Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer. J Biol Chem 273:3158–3165[Abstract/Free Full Text]
  19. Landau RL, Bergenstal DM, Lugibihl K, Kascht ME 1955 The metabolic effects of progesterone in man. J Clin Endocrinol Metab 15:1194–1215
  20. Landau RL, Lugibihl K 1958 Inhibition of the sodium-retaining influence of aldosterone by progesterone. J Clin Endocrinol Metab 18:1237–1245
  21. Wambach G, Higgins JR 1978 Antimineralocorticoid action of progesterone in the rat: correlation of the effect on electrolyte excretion and interaction with renal mineralocorticoid receptors. Endocrinology 102:1686–1693[Abstract/Free Full Text]
  22. Oparil S, Ehrlich EN, Lindheimer MD 1975 Effect of progesterone on renal sodium handling in man: relation to aldosterone excretion and plasma renin activity. Clin Sci Mol Med 49:139–147[Medline]
  23. Dunn JF, Nisula BC, Rodbard D 1981 Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 53:58–68[Abstract/Free Full Text]
  24. Zhang Y, Dufort I, Rheault P, Luu-The V 2000 Characterization of a human 20{alpha}-hydroxysteroid dehydrogenase. J Mol Endocrinol 25:221–228[Abstract]
  25. Nishizawa M, Nakajima T, Yasuda K, Kanzaki H, Sasaguri Y, Watanabe K, Ito S 2000 Close kinship of human 20{alpha}-hydroxysteroid dehydrogenase gene with three aldo-keto reductase genes. Genes Cells 5:111–125[Abstract]
  26. Penning TM, Burczynski ME, Jez JM, Hung CF, Lin HK, Ma H, Moore M, Palackal N, Ratnam K 2000 Human 3{alpha}-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem J 351:67–77[CrossRef][Medline]
  27. Luu-The V, Dufort I, Pelletier G, Labrie F 2001 Type 5 17ß-hydroxysteroid dehydrogenase: its role in the formation of androgens in women. Mol Cell Endocrinol 171:77–82[CrossRef][Medline]
  28. Suzuki-Yamamoto T, Nishizawa M, Fukui M, Okuda-Ashitaka E, Nakajima T, Ito S, Watanabe K 1999 cDNA cloning, expression and characterization of human prostaglandin F synthase. FEBS Lett 462:335–340[CrossRef][Medline]
  29. Miller WL, Auchus RJ, Geller DH 1997 The regulation of 17,20-lyase activity. Steroids 62:133–142[CrossRef][Medline]
  30. Swart P, Swart AC, Waterman MR, Estabrook RW, Mason JI 1993 Progesterone 16{alpha}-hydroxylase activity is catalyzed by human cytochrome P450 17{alpha}-hydroxylase. J Clin Endocrinol Metab 77:98–102[Abstract]
  31. Lin D, Black SM, Nagahama Y, Miller WL 1993 Steroid 17{alpha}-hydroxylase and 17,20-lyase activities of P450c17: contributions of serine106 and P450 reductase. Endocrinology 132:2498–2506[Abstract/Free Full Text]
  32. Auchus RJ, Miller WL 1999 Molecular modeling of human P450c17 (17{alpha}-hydroxylase/17,20-lyase): insights into reaction mechanism and effects of mutations. Mol Endocrinol 13:1169–1182[Abstract/Free Full Text]
  33. Thigpen AE, Silver RI, Guileyardo JM, Casey ML, McConnell JD, Russell DW 1993 Tissue distribution and ontogeny of steroid 5{alpha}-reductase isoenzyme expression. J Clin Invest 92:903–910
  34. MacDonald PC, Dombroski RA, Casey ML 1991 Recurrent secretion of progesterone in large amounts: an endocrine/metabolic disorder unique to young women? Endocr Rev 12:372–401[Abstract/Free Full Text]
  35. Concas A, Follesa P, Barbaccia ML, Purdy RH, Biggio G 1999 Physiological modulation of GABA (A) receptor plasticity by progesterone metabolites. Eur J Pharmacol 375:225–235[CrossRef][Medline]
  36. Li X, Bertics PJ, Karavolas HJ 1997 Regional distribution of cytosolic and particulate 5{alpha}-dihydroprogesterone 3{alpha}-hydroxysteroid oxidoreductases in female rat brain. J Steroid Biochem Mol Biol 60:311–318[CrossRef][Medline]
  37. Stoffel-Wagner B 2001 Neurosteroid metabolism in the human brain. Eur J Endocrinol 145:669–679[Abstract]
  38. Kumar D 1966 Tissue progesterone concentrations of placentas in pre-eclamptic patients. Am J Obstet Gynecol 95:594–595[Medline]
  39. Chesley LC, Tepper IH 1967 Effects of progesterone and estrogen on the sensitivity to angiotensin II. J Clin Endocrinol Metab 27:576–581[Abstract/Free Full Text]
  40. Brunette MG, Leclerc M 2002 Renal action of progesterone: effect on calcium reabsorption. Mol Cell Endocrinol 194:183–190[CrossRef][Medline]
  41. Reckelhoff JF, Zhang H, Srivastava K, Granger JP 1999 Gender differences in hypertension in spontaneously hypertensive rats: role of androgens and androgen receptor. Hypertension 34:920–923[Abstract/Free Full Text]
  42. Reckelhoff JF, Granger JP 1999 Role of androgens in mediating hypertension and renal injury. Clin Exp Pharmacol Physiol 26:127–131[CrossRef][Medline]
  43. Reckelhoff JF, Zhang H, Granger JP 1998 Testosterone exacerbates hypertension and reduces pressure-natriuresis in male spontaneously hypertensive rats. Hypertension 31:435–439[Abstract/Free Full Text]



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