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*PREDNISOLONE
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Endocrinology Vol. 139, No. 4 1533-1539
Copyright © 1998 by The Endocrine Society

ARTICLES

Reduced 11ß-Hydroxysteroid Dehydrogenase Activity in the Remaining Kidney Following Nephrectomy1

Geneviève Escher, Bruno Vogt, Thomas Beck, Daniel Guntern, Brigitte M. Frey and Felix J. Frey

Division of Nephrology, Department of Medicine, University Hospital of Berne, Inselspital, 3010 Berne, Switzerland

Address all correspondence and requests for reprints to: Felix J. Frey, Division of Nephrology, Freiburgstrasse 3, Inselspital, 3010 Berne, Switzerland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracellular access of steroids to gluco- and mineralocorticoid receptors is regulated by reduced 11ß-hydroxysteroid dehydrogenase (OHSD) 1 and 2. These enzymes convert active 11ß-OH-steroids into inactive 11-keto-steroids. The purpose of the present study was to establish whether the 11ß-OHSD1 and 11ß-OHSD2 are modulated in the remnant kidney 24 h or 14 days after uninephrectomy (UNX) in rats. Overall, 11ß-OHSD activity was analyzed by measuring the ratio of the exogenous 11ß-OH-steroid prednisolone to its 11-keto metabolite prednisone in vivo in kidney tissue using high performance liquid chromatography. To determine which isoenzyme accounts for the changed activity 24 h after UNX, the oxidation and reduction attributable to 11ß-OHSD1 and oxidation to 11ß-OHSD2 were analyzed in total renal extracts and in isolated glomeruli, proximal convoluted tubules (PCT), cortical ascending limbs, and cortical convoluted tubules (CCT). The messenger RNA content of 11ß-OHSD1 and 11ß-OHSD2 was measured by RT-PCR in renal tissues and single segments, using glyceraldehyde-3-phosphate-dehydrogenase as an internal standard. Protein amounts of 11ß-OHSD1 and 11ß-OHSD2 were assessed by Western blot. The prednisolone/prednisone ratio increased 24 h after UNX in 9 out of 10 animals (P <= 0.0011), and was unchanged 14 days after UNX. 11ß-OHSD1 oxidation (P <= 0.032) and reduction activity (P <= 0.002) declined 24 h after UNX in total extracts. 11ß-OHSD1 oxidase activity was more than 3 times higher in PCT than in glomeruli, cortical ascending limbs, and CCT, and declined by 50% after UNX (P <= 0.001). The reductase activity did not change following UNX in PCT. 11ß-OHSD2 activity was 5–15 times higher in CCT than in the other segments, and decreased significantly after UNX (P <= 0.008). UNX did not affect messenger RNA and protein levels of both enzymes in total renal extracts. In conclusion, 11ß-OHSD1 and 11ß-OHSD2 are predominantly expressed in PCT and CCT, respectively, and their corresponding oxidative activities decline after UNX. Thus, the access of 11ß-glucocorticosteroids to gluco- and mineralocorticoid receptors in the remaining kidney is facilitated after UNX.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLOWING uninephrectomy (UNX), a compensatory hypertrophy of the remaining kidney occurs. An increase in the RNA/DNA ratio and an increase in the protein/cell ratio in the absence of an augmentation in DNA synthesis are the relevant elements for the definition of cellular hypertrophy (1, 2). However the molecular events of the enhanced protein synthesis in hypertrophied cells have not been delineated in detail (1, 2, 3). Several factors including insulin-like growth factor, epidermal growth factors, phospholipase D, androgens, putative renotropic factors, and stress-related genes like heat shock protein 70 have been suggested to be involved in the compensatory hypertrophy of the kidney following UNX (4, 5, 6, 7, 8). Whatever it is that triggers the onset of compensatory hypertrophy of the kidney after UNX, a rapidly changing pattern of gene expression within the first 24 h, as assessed by measuring semiquantitatively the messenger RNA (mRNA) of an array of growth control elements, has been described (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Glucocorticosteroid molecules, together with their cognate receptor, act as potent transcription factors and modulate many of these growth control elements (11, 12). Thus modulation of the glucocorticoid effect following UNX might be relevant for the understanding of the compensatory renal hypertrophy.

The amount of glucocorticoids available in a given cell depends on two factors: first, the unbound steroid concentrations in the serum, and second, the intracellular availability of the steroid hormones. The unbound steroid concentrations in the serum are determined by the production and clearance rate of 11ß-OH-steroids (13), whereas the intracellular access of the steroid to the cognate receptor is regulated by the reduced 11ß-hydroxysteroid dehydrogenase (11ß-OHSD) enzymes, which convert biologically active endogenous and exogenous 11ß-OH-steroids into inactive 11-keto molecules (14, 15).

Two isoenzymes of 11ß-OHSD have been cloned (16, 17). 11ß-OHSD1 is NADP dependent and exhibits a lower affinity for the steroid molecules than 11ß-OHSD2, which is NAD dependent (17, 18). The main function of 11ß-OHSD2 is most likely the protection of the aldosterone receptor from promiscuous access of endogenous glucocorticoids to mineralocorticoid receptors (MRs) (19). Furthermore, there is evidence that 11ß-OHSD1 and/or 11ß-OHSD2 regulate access of glucocorticosteroids to the glucocorticoid receptors (GRs) (20, 21).

The aim of this study was to investigate the expression of 11ß-OHSD1 and 11ß-OHSD2 in the kidney of uninephrectomized rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Supplies
The steroids used for high performance liquid chromatography (HPLC) were purchased from Steraloids (Wilton, NH); organic solvents, HPLC grade, were from Rathburn (Walkerburn, Scotland); and the HPLC column was from Machery & Nagel (Oensingen, Switzerland). Hydeltrasol (prednisolone sodium phosphate) was obtained from MSD (Zuerich, Switzerland). For the measurement of 11ß-OHSD activity, corticosterone and 11-dehydrocorticosterone were obtained from Sigma (Buchs, Switzerland), and NADP, NADPH, and NAD were from Boehringer Mannheim (Rotkreuz, Switzerland). [1,2,6,7 3H]Corticosterone with a specific activity of 83 Ci/mM was purchased from Amersham (Aylesbury, Buckinghamshire, UK). [3H]11-Dehydrocorticosterone was prepared as already described (22). Bicinchonic acid protein assay reagent was from Pierce Chemical (Rockford, IL). Triton X-100 and thin-layer chromatography (TLC) plates (60 F 254) coated with silica gel were from Merck (Darmstadt, Germany). For RT-PCR, deoxynucleotide triphosphates (dNTPs), RNase inhibitor, avian myeloblastosis virus reverse transcriptase, and BSA were obtained from Boehringer Mannheim and Taq Gold polymerase from Perkin- Elmer (Rotkreuz, Switzerland). The collagenase Pan Plus (from Clostridium histolyticum) was a Serva product (Feinchemikalien, Heidelberg, Germany).

Animals
The protocol was approved by the ethics committee at our institution.Eight-week-old female Wistar rats weighing 190–210 g were kept in a temperature-, humidity-, and light (12 h on)-controlled room and maintained on a normal chow diet without fluid restriction. On the morning of the experiment, rats were separated into two groups and administered with 10 mg/kg Hydeltrasol ip and 60 mg/kg pentobarbital 1 h before UNX. Surgery was performed under aseptic conditions. After dissection of the capsule and ligation of the vessels and the ureter, the left kidney was removed and frozen in liquid nitrogen. To maintain hydratation and caloric intake after surgery, 5 ml 5% glucose was given sc. Postoperative analgesia was performed by administration of 0.1% Lidocaine to the wound and by the addition of Paracetamol (70 mg/kg) to the drinking water during the first 24 h after intervention. Twenty-four hours or 2 weeks after UNX, rats were injected a second time with 10 mg/kg Hydeltrasol and pentobarbital 1 h before being killed by an overdose of CO2. Blood samples (5 ml) were taken from the vena cava in a heparinized syringe. Plasma was separated by centrifugation and stored at -20 C. The remaining kidney was immediately frozen in liquid nitrogen and kept at -70 C. Tissues were powdered with mortar and pestle in a mixture of dry ice and acetone before being used for prednisolone and prednisone measurements and for 11ß-OHSD activity determination.

A third group of nine rats was nephrectomized as described above and killed 24 h later without injection of Hydeltrasol to analyze the mRNA and the protein of 11ß-OHSD1 and 11ß-OHSD2.

For analysis of single nephron segments, two groups of six rats were dissected for the enzymatic assays, and two rats of each group were used for RT-PCR.

Measurement of prednisolone and prednisone in plasma and tissues
Prednisolone and prednisone were measured in plasma and tissue by a method previously developed by us (22, 23, 24). The detection limit of prednisolone and prednisone was between 5 and 10 ng/ml or 5 and 10 mg of tissue.

Optimization of extraction procedure
Transfection studies with the complementary DNA (cDNA) of rat 11ß-OHSD1 and human 11ß-OHSD2 in COS-1 cells were performed, and the extraction and incubation procedure were optimized. Extraction buffers, incubation buffers, cofactor specificity, substrate concentrations, and Triton X-100 were the five parameters analyzed.

Assay for 11ß-OHSD1
The assay was performed as previously described by Monder et al. (25). Oxidation or reduction at C-11 was determined by measuring the rate of conversion of corticosterone to 11-dehydrocorticosterone in the presence of NADP or 11-dehydrocorticosterone to corticosterone in the presence of NADPH. Renal tissues were extracted with 10 mM Tris-HCl, pH 7.5; 5 mM EDTA, pH 8; 1% Triton X-100; 2 mM phyenylmethylsulfonylfluoride; and 100 µg total protein was used for the reaction. The assay was performed in 0.25 mM NADP or NADPH; 100 mM Tris, pH 8.3; 100 nCi [3H]corticosterone or [3H]11-dehydrocorticosterone; and 5 µM corticosterone or 11-dehydrocorticosterone. Samples were incubated for 1 h at 37 C, the reaction was stopped on ice, and steroids were extracted with 1 vol ethyl acetate. The organic layer was separated by centrifugation at 13,000 rpm and evaporated under a stream of nitrogen. The steroid residue was dissolved in 20 µl methanol containing a mixture of 20 µg each of unlabeled corticosterone and 11-dehydrocorticosterone. This was quantitatively transferred to thin-layer plates and developed in chloroform-methanol (90:10 vol/vol). The spots corresponding to the steroids were located under an UV lamp, cut out, transferred to scintillation vials, and counted in scintillation fluid in a Tri-Carb 2000 CA (Packard Instrument Company, Meriden, CT) fluid scintillation counter. Specific activity was expressed as micromoles of product formed per microgram protein per hour.

For activity measurements in single nephron segments, the assay was performed in a final volume of 10 µl and transferred without prior ethyl acetate extraction onto the TLC plate directly with the cold marker.

Assay for 11ß-OHSD2
The assay was performed as previously described by Albitson et al. (17). Homogenization of 50–200 mg kidney tissues for measurement of 11ß-OHSD2 activity was performed in homogenization buffer containing 250 mM sucrose and 10 mM Tris-HCl pH 7.5. Two hundred micrograms of protein extract was incubated for 1 h at 37 C with 1 mM NAD, 10 nM corticosterone, and 50 nCi [3H]corticosterone in 500 µl homogenization buffer. The subsequent steps were the same as those described for 11ß-OHSD1.

RT mRNA and PCR
Total RNA was extracted from kidney tissues following the guanidium thiocyanate method (26). The RNA concentration was determined by measuring the absorption at 260 nm, and its quality was controlled by loading 1 µg on a 1% formaldehyde gel.

RT was performed in 20 µl containing 50 mM Tris-HCl, pH 8.2; 6 mM MgCl2; 10 mM dithiothreitol; 100 mM NaCl; 200 µM dNTPs; 11 U RNAsin; 1 U avian myeloblastosis virus reverse transcriptase; 10 pmol 3' primer of the corresponding cDNA position [852–873 for 11ß-OHSD1, 1271–1295 for 11ß-OHSD2, 980-1004 for the internal standard glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), 2351–2371 for the GR, and 2865–2884 for the MR] and 2 µg total RNA (16, 17, 27, 28, 29, 30). Initially the 3' primer was incubated with total RNA for 5 min at 65 C and cooled at room temperature for 15 min. The remaining reaction components were added and incubated for 1h at 42 C.

PCR was performed in a total volume of 30 µl with 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 µM dNTPs; 10 pmol appropriate 3' and 5' cDNA primers (5' primer: 117–137 for 11ß-OHSD1, 381–406 for 11ß-OHSD2, 66–90 for GAPDH, 1838–1857 for GR, and 2504–2523 for MR); 6 µg BSA; 1 µCi [{alpha}-32P]deoxycytidine triphosphate; 2 µl reverse transcribed cDNA (diluted 10x for 11ß-OHSD1, undiluted for 11ß-OHSD2, diluted 5x for GAPDH); and 1 U Thermus aquaticus DNA polymerase. The mixture was overlaid with oil, and cDNA was amplified with a DNA thermal cycler (Perkin-Elmer Cetus) for 35 cycles. The amplification profile involved denaturation at 94 C for 1 min and 15 sec, primer annealing at 60 C for 2 min, and elongation of annealed primers at 72 C for 3 min. Ten microliters of each PCR reaction mixture were mixed with 2 µl 6-fold loading buffer and applied on a 0.9% agarose gel containing ethidium bromide. Electrophoresis was carried out with a constant voltage of 8 V/cm for 40 min. Bands were visualized under UV light and excised from the gel. Control experiments showed that by using 35 cycles, the technique was still in the linear range. The radioactivity was measured in a scintillation counter using a Cerenkov program.

For RT-PCR in single nephron segments, 6 µl RT material was used, and the number of cycles was increased to 45 for 11ß-OHSD1, GR, and MR and to 50 for 11ß-OHSD2 and GAPDH.

Western blot analysis
Electrophoresis was performed using a 12.5% polyacrylamide gel under reducing conditions. Prestained protein standards were used as markers. Probes were heated along with SDS sample buffer at 95 C for 15 min. After electrophoresis, the gel was equilibrated in transfer buffer (20 mM Tris, 190 mM glycine, 20% methanol) for 10 min. The transfer of protein to an Immobilon membrane (Millipore, Bedford, MA) was performed with a constant voltage of 60 V for 1 h on ice. The membrane was blocked in 5% BSA in PBS for 2 h, washed with PBS, and incubated overnight with a rabbit polyclonal antibody for 11ß-OHSD1 (1:10,000 in 0.5% BSA in TBS; a gift from Carl Monder, New York) and a rabbit polyclonal antibody for 11ß-OHSD2 (1:10,000 in 0.5% BSA in TBS; a gift from Zigmut Krozowski, Prahan, Australia). The Immobilon membrane was washed with PBS, saturated again for 2 h in 5% BSA, and incubated for 1 h at room temperature with a goat antirabbit IgG horseradish peroxidase conjugate (Bio-Rad Labs., Hercules, CA) diluted 1:5,000 in PBS containing 0.5% BSA. After washing, the detection was performed with the enhanced chemiluminescence (ECL) kit (Amersham).

Isolation of nephron segments
Rats with and without UNX were anesthetized with 10 mg/kg pentobarbital and perfused via the aorta with an ice-cold modified Hanks perfusion solution (137 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 0.33 mM Na2HPO4, 0.44 mM KH2PO4, 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose, 10 mM Tris-HCl, pH 7.4) followed by perfusion with a solution containing collagenase (2 mg/ml) in addition to the aforementioned solution. At the end of the perfusion, the kidney was removed, and thin pyramid pieces were incubated at 30 C for 20 min in a perfusion solution containing 0.5 mg/ml collagenase and 0.1 mg/ml BSA. Glomeruli, proximal convoluted tubules (PCT), cortical ascending limbs (CAL), and cortical collecting tubules (CCT) were isolated under a stereomicroscope as previously described (31, 32). Tubular length was measured using a millimeter scale placed under the microdissection dish. For the enzymatic assay, 1-mm-long segments or 2 glomeruli were used. For RT-PCR, RNA extraction was performed out of 10 segments or glomeruli, and the total RNA obtained was split for the five different reactions.

Transfection
Transfection with the cDNA of rat 11ß-OHSD1 and human 11ß-OHSD2 in COS-1 cells was performed using the diethylaminoethyl-dextran method (16, 17, 22, 33). Forty-eight hours after transfection, cells were harvested and extracted with two different buffers, one containing 10 mM Tris-HCl, pH 7.5; 5 mM EDTA; and 1% Triton DF-18 (a gift from C. Monder) (buffer A), and one containing 10 mM Tris-HCl, pH 7, and 25 mM sucrose (buffer B). After sonication, protein determination was performed using the bicinchonic acid reagent. For measuring the activity of 11ß-OHSD, 50 µg total protein from transfected cells was used. Triton extracts and sucrose extracts were incubated for 1 h at 37 C in buffer A or B in the presence of 1 mM NADP or NAD at two different substrate concentrations each (5 µM or 5 nM final concentration). Steroids were extracted, and TLC was performed as described above. The activity was expressed as percent of substrate converted to its dehydro form per hour in 50 µg total protein.

Analysis of data
Values are expressed as mean ± SD. Statistical differences were assessed by the paired t test and the Wilcoxon signed rank test. Corrections for multiple comparisons were performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prednisolone/prednisone ratios in kidney tissue
In each animal, the prednisolone/prednisone ratio was assessed on two occasions: first, before UNX in the kidney that was removed, and second, in the remaining kidney, at the time point when the animals were killed, i.e. 24 h or 2 weeks after UNX. Twenty-four hours after UNX, the prednisolone/prednisone ratio was higher in 9 out of 10 rats (1.3 ± 0.3 vs. 1.8 ± 0.7, mean ± SD; P < 0.011). The individual values are given in Fig. 1Go. In the group of animals killed 2 weeks after UNX, no significant difference of the ratio was observed in kidney tissues before and after UNX (1.3 ± 0.4 vs. 1.4 ± 0.2, mean ± SD; NS) (individual values not shown). The ratio of prednisolone/prednisone is concentration dependent (24, 34, 35). To exclude that the differences observed before and after UNX were attributable to this concentration dependency, prednisolone was also measured in plasma. Analysis of plots between prednisolone concentrations in plasma or tissue vs. prednisolone/prednisone ratios in kidney tissue revealed that the differences were not attributable to differences of prednisolone concentrations (data not shown).



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  		Figure 1. Impact of UNX on 11ß-OHSD activity assessed by distribution of prednisolone and prednisone in kidney tissues of rats. Prednisolone and prednisone were measured in removed kidney 1 h after injection of prednisolone. Twenty-four hours after UNX, the same measurements were performed in the remaining kidney. Each pair of points represents one animal (n = 10). Prednisolone/prednisone ratio increased in 9 out of 10 rats.

 
Optimization of extraction procedure
At the present time, 11ß-OHSD activity is attributed to two enzymes. First, 11ß-OHSD1 was described as the predominantly NADP-dependent enzyme that converts cortisol to cortisone (16, 36, 37). Later, 11ß-OHSD2, a high-affinity, NAD-dependent enzyme was cloned (17). Both enzymes are present in kidney tissues (17, 38). To distinguish between these two enzymes in an organ where both isoenzymes are present, we performed transfection studies with the cDNA of rat 11ß-OHSD1 and human 11ß-OHSD2 in COS-1 cells and optimized the extraction procedure. As shown in Table 1Go, COS-1 cells transfected with 11ß-OHSD1 could also convert corticosterone to 11-dehydrocorticosterone with NAD as a cofactor, when 5 nM corticosterone was used as a substrate and Triton added to the incubation and extraction buffers (11.8%, Table 1Go). The same nonspecificity of the cofactor was observed when the extraction and incubation were performed with sucrose (13.2%, Table 1Go). Using NADP as a cofactor and Triton for both the extraction procedure and the incubation, the 11ß-OHSD2 did not appear to significantly influence the activity of 11ß-OHSD1 (54.8% vs. 2.0% and 67.3% vs. 2.1% respectively; Table 1Go). A satisfactory specificity for 11ß-OHSD1 was obtained when sucrose was used for the extraction procedure and NADP together with Triton for the incubation (51.2% vs. 2% and 64.5% vs. 1.6%, respectively, Table 1Go). For the present investigation, it was decided to use Triton extraction with Triton buffer and NADP as a cofactor with 5 µM corticosterone as a substrate for measuring 11ß-OHSD1 (54.8%, Table 1Go). For the assessment of 11ß-OHSD2, the following conditions were used: 5 nM of corticosterone, NAD as a cofactor in a sucrose extract, and a sucrose buffer. This decision was based on the 49.6% conversion rate given in Table 1Go. By applying these conditions, 11ß-OHSD1 was still active (13.2%, Table 1Go). To abrogate the activity of 11ß-OHSD2, 1% of Triton, DF-18 was added to the assay (1.5%, Table 1Go). The differences between the values obtained with sucrose and Triton containing buffers—using sucrose extracted material for the incubation with NAD and 5 nM of corticosterone for both buffers—were considered as a measure for 11ß-OHSD2 activity.


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  		Table 1. Impact of substrate concentration, cofactors, and composition of incubation and extraction buffers on activity (% of conversion) of 11ß-OHSD1 and 11ß-OHSD2

 
Measurement of 11ß-OHSD activity in kidney extracts
Because prednisolone/prednisone ratios were not changed 2 weeks after UNX, detailed 11ß-OHSD1 and 11ß-OHSD2 activity measurements were performed only in samples 24 h after UNX. Twenty-four hours after UNX, the activity of 11ß-OHSD1 as assessed by the oxidation of corticosterone to 11-dehydrocorticosterone was decreased in 9 out of 10 animals (33.5 ± 9.2 vs. 26.9 ± 7.7% conversion of corticosterone to 11-dehydrocorticosterone per h/100 µg of total protein; P <= 0.032) (Fig. 2Go). The 11ß-OHSD1 reductase activity was also lower 24 h after UNX in 9 out of 10 rats (12.5 ± 2.9 vs. 10.5 ± 2.8% conversion of 11-dehydrocorticosterone to corticosterone per h/100 µg total protein; P <= 0.002). The reduction in reductase activity was very similar to the reduction of dehydrogenase activity (16% vs. 19%). The fact that the specific dehydrogenase activity reflecting 11ß-OHSD1 activity was higher than reductase activity is an established finding, and reflects the instability of the reductase in nature.



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  		Figure 2. Influence of UNX on oxidation of corticosterone (left), reduction of 11-dehydrocorticosterone (middle), and ratios of reduction/oxidation (right) in presence of NADP or NADPH as a cofactor and 5 µM corticosterone. Each pair of points represents one animal (n = 10). All values are expressed as percentage of substrate converted per 100 µg/total protein per hour and are the mean of three experiments. After UNX, a significant decrease in oxidation and reduction was observed. Calculated reduction/oxidation ratio did not increase significantly.

 
The activity of 11ß-OHSD2 measured in total kidney extracts remained unchanged following UNX (data not given).

Measurement of 11ß-OHSD activity in isolated segments of the nephron
11ß-OHSD1 and 11ß-OHSD2 activity were analyzed in glomeruli, PCT, CAL, and CCT (Fig. 3Go). 11ß-OHSD1 oxidase activity was more than 3 times higher in PCT than in glomeruli, CAL, and CCT in control animals (P <= 0.001) (Fig. 3Go, left). Twenty-four hours after UNX, 11ß-OHSD1 oxidase activity decreased significantly in PCT (11.3 ± 2.5 vs. 5.6 ± 2.7, P <= 0.001), whereas no significant changes were observed in the other segments. 11ß-OHSD1 reductase activity was higher in PCT than in the other segments in control rats (P <= 0.018) (Fig. 3Go, middle). This activity decreased in CAL (10.5 ± 3.7 vs. 6.5 ± 2.3, P <= 0.036), increased in PCT (13.9 ± 1.2 vs. 17.3 ± 2.9, P <= 0.01), and remained unchanged in the other segments following UNX (Fig. 3Go, middle). 11ß-OHSD2 activity was found mainly in CCT, where it was 5–15 times higher than in the other segments (Fig. 3Go, right). In CCT, the activity of 11ß-OHSD2 decreased after UNX from 64.3 ± 19.8 to 44.7 ± 18.8, P <= 0.008 (Fig. 3Go, right). To demonstrate that the activity measurements were attributable to 11ß-OHSD2, Triton X-100 was added to the CCT before the measurements were performed. Triton decreased the activity from 64.3 ± 19.8 to 3.5 ± 1.6 and from 44.7 ± 18.8 to 5.6 ± 2.6, respectively. The post Triton X-100 values were close to the background values.



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  		Figure 3. Effect of UNX on 11ß-OHSD1 and 11ß-OHSD2 activity in isolated segments of nephron. Solid columns, Control animals; hatched columns, UNX animals. When the same enzymatic reactions from various segments were compared, 11ß-OHSD1 activity was highest in PCT, whereas that of 11ß-OHSD2 was highest in CCT. Oxidase activity of 11ß-OHSD1 and 11ß-OHSD2 declined significantly following UNX in PCT and CCT, respectively.

 
Measurement of steady-state mRNA in total kidney extracts and isolated segments of nephron
For mRNA analysis in total extracts, a group of nine animals was studied 24 h after UNX. After extraction of total RNA from kidney tissue, the quantification of mRNA was performed by RT-PCR using specific primers for 11ß-OHSD1 and 11ß-OHSD2. As internal standard, GAPDH was used (Fig. 4Go). The incorporation of [{alpha}-32P]deoxycytidine triphosphate in 11ß-OHSD1 and 11ß-OHSD2 was not significantly decreased 24 h after UNX when standardized with GAPDH (11ß-OHSD1: 1.73 ± 0.34 vs. 1.36 ± 0.34, NS; 11ß-OHSD2: 1.64 ± 0.41 vs. 1.30 ± 0.41, NS).



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  		Figure 4. Agarose gel electrophoresis of PCR products of 11ß-OHSD1, 11ß-OHSD2, and GAPDH in control kidney and in kidney tissue 24 h after UNX. Total RNA (2 µg) was reverse transcribed using appropriate 3' primers. For 11ß-OHSD1 mRNA 20 ng, for 11ß-OHSD2 200 ng, for GAPDH 100 ng of RNA after reverse transcription was used as a template for PCR reactions. M, Molecular weight marker ({lambda}/HindIIIEcoRI); 1, PCR blank (no cDNA); 2, kidney before UNX; 3, kidney 24 h after UNX.

 
To get an estimate of the transcription in the isolated segments, a restricted number of semiquantitiative measurements were performed. When the mRNA of 11ß-OHSD1 in isolated segments of the nephron (glomeruli, CCT, CAL, and PCT) were analyzed in two rats of each group, no significant changes were observed. The mRNA of 11ß-OHSD2 was detectable only in the CCT, and its level not affected by UNX. The mRNA of the GR (in glomeruli, PCT, CAL, and CCT) and of the MR (in CCT and CAL) also remained unchanged.

Measurement of 11ß-OHSD protein
The amount of 11ß-OHSD1 and 11ß-OHSD-2 protein was measured in the second group of nephrectomized rats by Western blot analysis using the appropriate polyclonal antibody (Fig. 5Go). For 11ß-OHSD1 and 11ß-OHSD2 protein, the relative transmission measured remained unchanged after UNX (8.7 ± 3.9 vs. 10.4 ± 4.8 relative transmission per 50 µg total protein, NS; and 4.6 ± 0.9 vs. 5.0 ± 1.8 relative transmission per 50 µg total protein, NS, respectively).



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  		Figure 5. Western blot analysis of 11ß-OHSD1 and 11ß-OHSD2 protein from kidney before and 24 h after UNX. Fifty micrograms protein from tissue homogenates in buffer A were used for 11ß-OHSD1 protein measurement and 50 µg tissue homogenate in buffer B was taken to analyze 11ß-OHSD2 protein. 1, before UNX; 2, 24 h after UNX.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation revealed a significant increase in the ratio of prednisolone/prednisone in kidney tissue following UNX, when exogenous prednisolone was administered. As recently shown, this ratio reflects the activity of 11ß-OHSD in vivo (22, 24). The increased ratio of the 11ß-hydroxysteroid to the corresponding 11-ketosteroid is determined by the activity of 11ß-OHSD1 and 11ß-OHSD-2 present in the kidney. Both enzymes have been cloned, and it has been shown that 11ß-OHSD1 is NADP dependent and 11ß-OHSD2 is NAD dependent (16, 17). Thus the question arises whether UNX modifies the activity of 11ß-OHSD1 or 11ß-OHSD2.

To distinguish between the activity of 11ß-OHSD1 and 11ß-OHSD2 in tissue extracts, the cofactor dependency might be used as a discriminating factor. However analyzing the differential effect of NAD and NADP on the 11ß-OHSD activity did not allow us to distinguish specifically between the two enzymes in a given tissue. To increase the specificity of the measurements, we applied two additional extraction procedures. Monder et al. (37) used Triton X-100 as a detergent for extracting 11ß-OHSD out of tissues. The activity they obtained corresponded most likely to 11ß-OHSD1, an enzyme that was not cloned at that time. Albitson et al. (17), who cloned 11ß-OHSD2, extracted the enzyme with a buffer containing sucrose. As shown in Table 1Go, the two different extraction procedures allowed us to increase the specificity of the measurements, because Triton X-100 virtually completely destroyed 11ß-OHSD2. The effect of Triton X-100 on 11ß-OHSD2 can be used to abrogate the activity of 11ß-OHSD2 in a mixture of 11ß-OHSD1 and 11ß-OHSD2 obtained following sucrose extraction. Thus by combining the three parameters of extraction procedure, cofactors, and composition of the incubation buffer, it was possible to assess specifically the activities of 11ß-OHSD1 and 11ß-OHSD-2. Note that the problem of distinguishing between 11ß-OHSD1 and 11ß-OHSD-2 exists only for the oxidative activity, because Krozowski et al. (17) showed that 11ß-OHSD2 is a unidirectional oxidative enzyme (17). Therefore, the total reductase activity was by and large exclusively due to 11ß-OHSD1, as shown by activity measurements in COS cells transfected with cDNA of 11ß-OHSD1 and 11ß-OHSD-2.

To demonstrate whether UNX modifies the activity of 11ß-OHSD1 or 11ß-OHSD2, we took advantage of the heterogenous distribution of these isoenzymes within the nephron. Using immunohistochemistry and in situ hybridization, it was shown that 11ß-OHSD1 was mainly located in the proximal convoluted tubule, whereas 11ß-OHSD2 was localized in the distal tubule (17, 18, 38). Furthermore, our group has demonstrated that 11ß-OHSD1 is expressed in glomerular mesangial cells (21). In the present investigation, we provide definite information about the topographic distribution of the 11ß-OHSD isoenzymes within tubular segments by analyzing for the first time mRNA and activity in dissected glomeruli, PCT, CAL, and CCT. The analysis revealed that PCT is harboring mainly 11ß-OHSD1 and CCT 11ß-OHSD2. Following UNX, the oxidation reaction of 11ß-OHSD1 declined, whereas the reduction increased in PCT and 11ß-OHSD2 oxidative activity decreased in CCT. Because these two tubular fragments account quantitatively for the largest part of the nephron, it is reasonable to conclude that the reduced overall activity of 11ß-OHSD in kidney tissue in vivo as assessed by the prednisolone/prednisone ratio is attributable to a decline of the oxidases in PCT and CCT and to the enhanced reductase in PCT.

Whereas the present study provides strong evidence with respect to the decreased activities of 11ß-OHSD isoenzymes following UNX, it does not provide conclusive evidence about the mechanism accounting for the decreased activity. The decreased activity cannot be explained by a decreased transcription or translation rate. Thus one might assume changes of the posttranslational modifications (39) and/or the presence of endogenous inhibitors. It is also possible that the in vivo change could be due to changes in cofactor availability or to changes in the elimination of the reduced and oxidized products or other factors not related to the enzyme activity. Alternatively, our approach to assess transcription and translation might not have been sensitive enough to depict the rapidly changes induced by UNX (1, 9, 40). There are other examples in which changes in the activity and transcription did not proceed in parallel (1, 41).

By determining in vivo the ratio between the biologically active 11ß-hydroxysteroid and its 11-keto metabolite, which is deficient of gluco- or mineralocorticoid activity, we have clearly established that following UNX the remaining kidney is in the initial phase exposed to an increased concentration of 11ß-hydroxysteroids. The biological relevance of such an increased exposure to 11ß-hydroxysteroids is open to speculation. First, during compensatory renal hypertrophy, which occurs within the first hour and days after removal of one kidney, an increased transcription of some but not all genes has been found (1, 42). GRs and MRs act as transcription factors when they bind to their cognate ligand. Because it is well established that 11ß-OHSD regulates locally the access of these ligands, i.e. of the 11ß-hydroxysteroids to both GRs and MRs, changes in the activity of 11ß-OHSD affect the pattern of gene expression. Second, the fractional urinary excretion of potassium increases with declining glomerular filtration rate. The mechanisms involved in this renal adaptation have been partially defined. Among other factors, aldosterone and Na+-K+-ATPase in the outer medulla have been suggested to play a role for such an adaptation (43, 44, 45). Fine et al. (46) indicated that the renal adaptation is an inherent characteristic of the renal tubular cells, although it remains unclear what is the exact mechanism for such an adaptation. There is some evidence that the Na+-K+-ATPase is enhanced by glucocorticoids, and that the activation of the MR by glucocorticoids is enhanced by an inhibited activity of 11ß-OHSD1 (32, 47, 48). Thus the decreased activity of 11ß-OHSD1 observed following UNX in the present investigation adds to the understanding of tubular adaptation associated with nephron reduction.


   Footnotes
 
1 This work was supported by a grant from the Swiss National Foundation for Scientific Research (No. 3200–040492.94). Back

Received September 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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