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(Hypertension. 1998;32:1077-1082.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Renal 11ß-Hydroxysteroid Dehydrogenase in Genetically Salt-Sensitive Hypertensive Rats

Yoshiyu Takeda; Satoru Inaba; Kenji Furukawa; Isamu Miyamori

From the Second Department of Internal Medicine (Y.T., S.I., K.F.) and Department of Health Sciences (Y.T.), School of Medicine, Kanazawa University, and Third Department of Internal Medicine, Fukui Medical School (I.M.), Japan.

Correspondence to Yoshiyu Takeda, MD, Second Department of Internal Medicine, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa 920, Japan. E-mail takeday{at}mhs.mp.kanazawa-u.ac.jp

	Abstract

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Abstract—Renal 11ß-hydroxysteroid dehydrogenase II (11ß-HSDII) converts glucocorticoids into inactive metabolites and plays an important role in controlling blood pressure and sodium retention. To examine whether this enzyme may be involved in the pathophysiology of salt-sensitive hypertension, we determined 11ß-HSDII activity and mRNA levels in the blood vessel and kidney of Dahl Iwai salt-sensitive (DS) rats and Dahl Iwai salt-resistant (DR) rats. Urinary free corticosterone:free 11-dehydrocorticosterone ratio was measured to estimate renal 11ß-HSD activity. Vascular 11ß-HSDII activity was expressed as percent conversion of [³H]corticosterone to [³H]11-dehydrocorticosterone in homogenized mesenteric arteries. 11ß-HSDII mRNA was estimated with the use of competitive polymerase chain reaction (PCR). Renal 11ß-HSDII activity and mRNA levels were significantly decreased in 8- and 12-week-old high salt DS rats compared with DR, Sprague-Dawley (SD), or low salt DS rats of the same age. Decreased 11ß-HSDII activity and mRNA levels in mesenteric arteries were observed in 8- and 12-week-old high salt DS rats. Urinary excretion of 11ß-HSDII inhibitory factors was measured by inhibition of enzyme activity in microsomes from human kidney. The urinary inhibitors were significantly increased in 8- and 12-week-old high salt DS rats compared with DR, SD, or low salt DS rats of the same age. There were no significant differences in 11ß-HSDII activity and mRNA levels in mesenteric arteries and kidney or in urinary inhibitors between 4-week-old DS, DR, and SD rats. These results indicate that 11ß-HSDII may play a role in salt sensitivity and development of hypertension in the DS rat.

Key Words: glucocorticoids • mineralocorticoids • rats, Dahl • kidney • hypertension, essential • sodium

	Introduction

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The enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) catalyzes the conversion of glucocorticoids to their inactive metabolites. A deficiency of 11ß-HSD, whether congenital or produced by inhibition of this enzyme related to the administration of licorice or carbenoxolone, leads to the activation of mineralocorticoid receptors by glucocorticoids, resulting in sodium retention and hypertension. Decreased 11ß-HSD activity has been demonstrated in some patients with essential hypertension^{1 2 3} and in rats with genetic hypertension.^{4 5}

Biochemical studies have revealed the existence of 2 isoforms of 11ß-HSD: NAD⁺ dependent and NADP⁺ dependent. 11ß-HSDII (the NAD⁺-dependent isoform) is found in distal portions of the nephron, where it has been shown to colocalize with mineralocorticoid receptors.⁶ Progesterone derivatives, which are potent inhibitors of 11ß-HSDII, have been shown to be active in conferring mineralocorticoid Na⁺-retaining activity and elevating blood pressure.^{7 8} We and others have previously shown that human urine contains substances that inhibit 11ß-HSDII, and these substances are elevated in subgroups of hypertension.^{9 10}

Excess sodium intake is intimately involved in the pathogenesis of hypertension. In large populations, significant correlations between the level of salt intake, blood pressure, and the frequency of hypertension have been reported. Since most people in Western countries, including Japan, ingest a high sodium diet, the fact that only about half will develop hypertension suggests a variable degree of blood pressure sensitivity to sodium, although obviously heredity and interaction with other environmental exposures may be involved.¹¹ Dahl salt-sensitive (DS) rats are widely used to study genetic determinants of salt-sensitive hypertension. In this strain, supplemental dietary sodium increases blood pressure, whereas in the Dahl salt-resistant (DR) strain, supplemental dietary sodium has little or no effect on blood pressure. There are several reports of the abnormalities of the renin-angiotensin system,¹² adrenal steroids,¹³ and sympathetic nerve system¹⁴ in DS rats. Recently, mutations of the gene for 11ß-hydroxylase, an adrenal enzyme involved in the synthesis of 18-hydroxy-11-deoxycorticosterone, in DR rats were reported.¹⁵ These mutations may not cause hypertension in DS rats because no mutations were found in DS rats or in Sprague-Dawley (SD) rats. Cover et al¹⁶ have reported abnormalities of the aldosterone synthase gene in DR rats. These abnormalities were not found in either DS or SD rats. These findings may not explain the cause of salt-sensitive hypertension in DS rats. To clarify the mechanism of salt-induced hypertension in DS rats, we compared 11ß-HSDII activity, gene expression of 11ß-HSDII in mesenteric arteries and kidneys, and urinary excretion of 11ß-HSDII inhibitory factors between DS, DR, and SD rats.

	Methods

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Male DS, DR, and SD rats (Eisai Supply, Eisai Animal Research Center), aged 3 to 4 weeks, were initially fed a standard chow (0.45% NaCl) purchased from Nippon Charles River. Both DS and DR rats were fed high sodium chow (7%) for 4 weeks (n=10 in each group) and for 8 weeks (n=10 in each group). SD rats were also fed high sodium chow (7%) for 4 weeks (n=10) and for 8 weeks (n=10). DS rats were fed standard chow for 8 weeks (n=10) and for 12 weeks (n=10). All rats were housed in metabolic cages, and daily urinary excretion was collected. The blood pressure was determined by the plethysmographic tail-cuff method, as previously reported.¹⁷ Blood was collected from the tail vein, as previously reported.¹⁷ Plasma concentrations of corticosterone and aldosterone were estimated by radioimmunoassay after purification of the plasma extracts by high-performance liquid chromatography (HPLC), as previously reported.¹⁸

Rat mesenteric arteries were removed immediately after decapitation under pentobarbital anesthesia and were placed in ice-cold 0.9% NaCl. The tissues were homogenized, and the microsomal fractions were prepared as described previously¹⁹ and assayed for protein colorimetrically (BioRad Laboratories).

The protocol was approved by the Animal Research Committee of the School of Medicine, Kanazawa University.

Measurements of Renal 11ß-HSDII Activity
Urinary free corticosterone:free 11-dehydrocorticosterone ratio was measured to estimate renal 11ß-HSD activity, as previously reported.²⁰ For the extraction of urinary free steroids, 1 mL of urine containing [³H]corticosterone (3000 cpm, Amersham Japan) or [³H]11-dehydrocorticosterone was passed through a prewashed (5 mL methanol, 10 mL water) Sep-Pak C18 cartridge (Waters). After the cartridge was washed with 10 mL water, steroids were eluted with 2x 3 mL methanol. The combined eluates were evaporated to dryness, redissolved in 40% methanol, and chromatographed in a reversed-phase HPLC system,⁴ followed by radioimmunoassay and individual recovery measurements. [³H]11-dehydrocorticosterone was synthesized in vitro by incubation of rat kidney NRK-52E cells (Dai Nippon Seiyaku, Tokyo, Japan) with 50 µCi [³H]corticosterone for 24 hours in Dulbecco's modified Eagle's medium at 37°C, as previously reported.²¹ Antibodies of corticosterone and 11-dehydrocorticosterone were purchased from Cosmo Bio Corp.

Measurements of Vascular 11ß-HSDII Activity
11ß-HSDII activity in mesenteric arteries was determined by measuring the rate of conversion of [³H]corticosterone to [³H]11-dehydrocorticosterone at 37°C for 30 minutes. The reaction mixture contained 20 µL of microsomal fraction of mesenteric arteries (250 µg protein), 10 µL of 1.12x10^-8 mol/L [³H]corticosterone, 250 µmol/L of NAD⁺, and 60 nmol/L of corticosterone, as previously reported.²² The reaction was stopped by adding volumes of ethyl acetate. Metabolites of corticosterone were separated by HPLC as mentioned above.

Measurements of Urinary 11ß-HSDII Inhibitory Factors
For determination of the retention time of 11ß-HSDII inhibitory factors, urine extracts were diluted with methanol to a final concentration of 30% methanol and chromatographed on a C18 Ultrasphere ODS column (5 µm, Beckman Instruments). Components were eluted with a methanol gradient beginning with 30% aqueous methanol that increased linearly to 100% methanol by 60 minutes at a flow rate of 1 mL/min. Each fraction was evaporated under nitrogen gas and assayed for inhibitory activity in 11ß-HSDII radioenzymatic assays. Radioenzymatic assay of urinary 11ß-HSDII inhibitory activity was performed by a procedure based on previously described methods.⁹ Briefly, human renal cortex microsomes (250 µg protein) were incubated at 37°C for 30 minutes with 1.12x10^-8 mol/L [³H]corticosterone and 250 µmol/L of NAD⁺ in 50 mmol/L Tris-HCl buffer (pH 8.5) in a total volume of 0.25 mL. For the assay, an aliquot of either water (control), urine sample (not hydrolyzed) that were major peak separated by HPLC, or 0.5% aqueous ethanol solution of glycyrrhetinic acid (GA) was added. The reaction was terminated by the addition of 4 mL of ethyl acetate. Metabolites of corticosterone were separated by HPLC as mentioned above, and the percentage of conversion of corticosterone to 11-dehydrocorticosterone was calculated. The percent inhibition was calculated relative to picomoles of GA (GA equivalence units) with the appropriate GA standard curve.

Competitive Polymerase Chain Reaction Assay of Renal 11ß-HSDII mRNA
Rat kidneys and mesenteric arteries were removed immediately after decapitation with the animals under pentobarbital anesthesia and were frozen in liquid nitrogen and stored at -80°C before use. Total RNA from rat renal cortex and mesenteric arteries was isolated with guanidine thiocyanate, followed by centrifugation in a cesium chloride solution.²³ One microgram of total RNA was incubated at 42°C for 60 minutes with 2.5 U M-MLV reverse transcriptase (RT) (Perkin-Elmer Japan) in a 20-µL reaction mixture containing random hexanucleotide primers. After incubation for 5 minutes at 99°C, the single-stranded cDNA in the 20-µL reaction mixture was amplified with a polymerase chain reaction (PCR) mixture containing 0.2 mmol/L of each dNTP. The reaction was followed by incubation at 92°C for 3 minutes and 30 cycles of the following sequential steps: 92°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes.

The sequences of sense and antisense primers for 11ß-HSDII were 5'-ACTCCGTGGCC-TGAGACG-3' and 5'-TTCAAGTCCACCACACAG-3', respectively, as previously described.²⁴ The sense and antisense primers for 11ß-HSDII correspond to nucleotides 1208 to 1227 and 1503 to 1522, respectively, of the complementary DNAs. The competitive templates for 11ß-HSDII were made with the use of the PCR MIMIC Construction Kit (Clontech), as previously reported.²⁴ After quantification, a set of serial dilutions was used as an internal standard for competitive PCR. Competitive PCR was performed with 2.5 µL of the reverse-transcribed DNA, 2 µL of different concentrations of the competitive template, 0.5 µmol/L each of sense and antisense primers, and 0.5 U of Taq DNA polymerase (Perkin-Elmer Japan) in 50 µL of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mol/L MgCl₂, and 0.2 mmol/L of each dNTP. The reactions were performed for 1 minute at 94°C, 1 minute at 59°C, and 2 minutes at 72°C for 30 cycles. Ten-microliter aliquots of amplification products were electrophoresed on a 3.0% agarose gel. The gel was stained with ethidium bromide and photographed.

The signal intensity was quantified by computerized densitometry with the BIO-PROFIL BIO-1D system (Compak). The intensities of each product from cDNA and from competitive templates were plotted as a function of the known amounts of the competitive templates. To test the yield and the efficiency of the reverse transcriptase reaction, 1 µg of total RNA was subjected to reverse transcription as above, with 5 µmol/L of radioactively labeled [³²P]dCTP (New England Nuclear) added to the reaction. The total volume of the RT reaction was increased to 30 µL. Before the addition of enzyme, 1 µL of the reaction was removed for the determination of trichloroacetic acid (TCA)–precipitable counts (background). After 1 hour of incubation at 42°C, 1 µL of the reverse transcription was taken out for measurement of incorporated labeled dCTP. Samples were precipitated in cold 5% TCA and filtered on Whatman GF/C fiberglass filters (Whatman Inc) under a slight vacuum. Filters were dried and placed in scintillation vials. After addition of scintillation fluid, samples were counted in a ß-counter. The amount of DNA synthesized was calculated by multiplying the fraction of total dCTP incorporated into TCA-precipitated counts per minute by the number of nanomoles of each dNTP in the reaction and the average weight of all 4 dNTPs. The intra-assay and interassay variabilities of the competitive PCR were 11.5% and 14.8%, respectively. The concentration of 11ß-HSDII mRNAs was expressed as attomoles per 100 ng of RNA.

The RT-PCR products in 10-µL aliquots were electrophoresed on a 3% agarose gel and transferred to nylon membranes. The membranes were prehybridized in 50% formamide, 5x SSC (1x SSC: 0.15 mol/L NaCl, 0.015 mol/L sodium citrate), 5x Denhardt's reagent, 1% SDS, and 0.5 g/L salmon sperm DNA at 50°C for 6 hours. They were then hybridized in the same buffer at 50°C for 15 hours with the specific oligoprobe for 11ß-HSDII (5'-GCCATCATTGATGCACTGCT-3') that had been end-labeled with [³²P]ATP (6000 Ci/mmol, New England Nuclear) with a 5'-end oligonucleotide labeling kit. Next, the membrane was washed twice in 2x SSC/0.1% SDS at room temperature for 20 minutes and twice in 0.1x SSC/0.1% SDS at 50°C for 20 minutes in preparation for autoradiography.

Data are expressed as mean±SEM. The significance of differences was assessed by 1-way ANOVA and a multiple comparison test. Statistical significance was accepted for P<0.05.

	Results

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Renal 11ß-HSDII inhibitory activity, as determined by HPLC analysis, peaked at 25 to 29 minutes (Figure 1). The standards for cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, deoxycorticosterone, and progesterone were eluted at the different retention times of this peak.

View larger version (20K): [in this window] [in a new window]   Figure 1. HPLC profile shows renal 11ß-HSDII inhibitory factors (11ß-HSDIF).

The Table shows body weight, systolic blood pressure, heart rate, plasma sodium and potassium, and plasma corticosterone and aldosterone concentrations of DS, DR, and SD rats. The blood pressure of 8- and 12-week-old DS rats on a high salt diet was significantly higher than that of DR, SD, or DS rats on a low salt diet of the same age (P<0.05). The plasma potassium and sodium concentrations did not differ between experimental groups. Plasma aldosterone concentrations were significantly lower in DS rats on a high salt diet than in DR, SD, or DS rats on a low salt diet (P<0.05). Plasma corticosterone concentrations did not show any significant differences between groups. Urinary free corticosterone:free 11-dehydrocorticosterone ratio was significantly higher in 8- and 12-week-old DS rats on a high salt diet than in SD, DR, or DS rats on a low salt diet of the same age (P<0.05) (Figure 2). There were no significant differences in these parameters between 4-week-old DS, DR, and SD rats. 11ß-HSDII activity in mesenteric arteries of 8- and 12-week-old DS rats on a high salt diet was significantly decreased compared with DR, SD, or DS rats on a low salt diet of the same age (P<0.05) (Figure 3). Specific mRNA for 11ß-HSDII could be detected in rat renal cortex and mesenteric artery by PCR analysis. Figure 4 shows that increasing concentrations of each competitive template for 11ß-HSDII from 0 to 80x10^-3 attomoles per microliter increasingly inhibited the amplification of endogenous 11ß-HSDII in kidney. Renal 11ß-HSDII mRNA levels in 8- and 12-week-old DS rats on a high salt diet were significantly lower than those in SD, DR, or DS rats on a low salt diet of the same age (P<0.05) (Figure 2). There were no significant differences in 11ß-HSDII mRNA levels between 4-week-old SD, DS, and DR rats. The concentration of 11ß-HSDII mRNA in mesenteric arteries of 8- and 12-week-old DS rats on a high salt diet was significantly lowered compared with DR, SD, or DS rats on a low salt diet of the same age (P<0.05) (Figure 3). The urinary excretion of the endogenous 11ß-HSDII inhibitory factor(s) was significantly increased in 8- and 12-week-old DS rats on a high salt diet compared with SD, DR, or DS rats on a low salt diet of the same age (P<0.05) (Figure 5). The urinary excretion of the inhibitory factor(s) did not differ between 4-week-old DS, DR, and SD rats. We measured urinary excretion of 11ß-HSDII inhibitory factor(s) using whole urine extracts, as previously reported.¹⁰ The urinary excretion of the inhibitory factor(s) from whole urine extracts was also increased in DS rats on a high salt diet (data not shown).

View this table: [in this window] [in a new window]   Table 1. Body Weight, Systolic Blood Pressure, Heart Rate, Serum Electrolytes, and Plasma Aldosterone and Corticosterone Concentrations in Experimental Rats

View larger version (46K): [in this window] [in a new window]   Figure 2. Renal 11ß-HSDII activity (urinary free corticosterone [B]:free 11-dehydrocorticosterone [A] ratio) (top) and concentrations of 11ß-HSDII mRNA (bottom) in SD, DR, and DS rats. DS (low salt) indicates DS rats on a low salt diet. *P<0.05 vs 8- and 12-week-old SD, DR, and DS rats on a low salt diet or 4-week-old SD, DR, and DS rats.

View larger version (49K): [in this window] [in a new window]   Figure 3. 11ß-HSDII activity (top) and concentrations of 11ß-HSDII mRNA (bottom) in mesenteric arteries of SD, DR, and DS rats. B indicates corticosterone; A, 11-dehydrocorticosterone; and DS (low salt), DS rats on a low salt diet. *P<0.05 vs 8- and 12-week-old SD, DR, and DS rats on a low salt diet or 4-week-old SD, DR, and DS rats.

View larger version (16K): [in this window] [in a new window]   Figure 4. Analysis of relative changes in 11ß-HSDII mRNA concentrations by competitive PCR. Increasing the concentration of competitive template for 11ß-HSDII from 0 to 80x10-3 attomoles per microliter increasingly inhibited the amplification of endogenous 11ß-HSDII cDNA in the kidney or mesenteric artery.

View larger version (40K): [in this window] [in a new window]   Figure 5. Urinary excretion of endogenous 11ß-HSDII inhibitory factor(s) (11ß-HSDII IF) in SD, DR, and DS rats. DS (low salt) indicates DS rats on a low salt diet. *P<0.05 vs 8- and 12-week-old SD, DR, and DS rats on a low salt diet or 4-week-old SD, DR, and DS rats.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The effect of aldosterone on sodium absorption has been studied in classic mineralocorticoid-sensitive tissues such as kidney, colon, and salivary glands. Mineralocorticoid receptors and 11ß-HSDII mRNA are similarly distributed in human and rat kidney.⁶ The crucial physiological role of 11ß-HSDII in conferring aldosterone selectivity on mineralocorticoid receptor in epithelial tissues has been amply demonstrated by the recent finding that patients with the syndrome of apparent mineralocorticoid excess (AME) have point mutations or deletions in the 11ß-HSDII gene, resulting in diminished or absent function.^{25 26}

Urinary free cortisol:free cortisone ratio is reported to be a sensitive index of renal 11ß-HSDII activity in humans.²⁰ We estimated this ratio as renal 11ß-HSDII activity in rat. Our study showed that urinary free corticosterone:free 11-dehydrocorticosterone ratio in DS rats was increased compared with DR or SD rats. The levels of 11ß-HSDII mRNA in the kidney were lower in DS rats than in DR or SD rats. These results indicate that renal 11ß-HSDII activity is decreased in DS rats. Decreased renal 11ß-HSDII activity may play a role in salt sensitivity and development of hypertension in DS rats. However, in rats 11ß-HSDI in kidney is more abundant than 11ß-HSDII.²⁷ Brem et al²⁸ reported that a high sodium diet increases 11ß-HSDI activity and levels of mRNA in normal dog kidney but does not change 11ß-HSDII activity and levels of mRNA. Fanco-Saenz et al²⁹ demonstrated that hypertensive DS rats (aged 10 weeks) had lower kidney 11ß-HSD. In our data, plasma potassium concentrations in DS rats did not differ from those in DR or SD rats. Patients with AME show hypokalemia; however, concentrations of plasma potassium in AME range from 0.9 to 3.8 mmol/L and do not correlate with the activity of 11ß-HSD.²⁵ Recently, normokalemic AME with abnormal 11ß-HDSII gene was reported.³⁰ Normokalemic primary aldosteronism has also been reported.^{31 32} Walker et al² reported that half-time periods of 11-[-H³]cortisol were prolonged in a subgroup of hypertensive patients who did not show hypokalemia. Thus, a mineralocorticoid excess state does not always show hypokalemia. In our data, plasma aldosterone levels were reduced in DS rats. Patients with AME also show low aldosterone levels.²⁵ Micropuncture studies examining segmental NaCl transport in Dahl rats have demonstrated no differences in NaCl transport beyond the loop segment between S and R strains. Kudo et al³³ reported that cultures from the cortical collecting duct of DS rats show no transport abnormalities compared with cultures from DR rats. DS rats with a low sodium diet did not show hypertension and had no decreased renal 11ß-HSDII activity and mRNA levels. Taken together, there is a possibility that reduced 11ß-HSDII activity in kidney of DS rats may be a consequence rather than a cause of hypertension. However, 11ß-HSDII activity and mRNA levels in blood vessels were decreased in hypertensive DS rats. Smith et al³⁴ reported the presence of 11ß-HSDII in vascular smooth muscle cells by immunohistochemistry. We detected the expression of 11ß-HSDII mRNA in cultured vascular smooth muscle cells using RT-PCR methods (data not shown).

There has been increasing evidence that mineralocorticoids, acting on peripheral vascular tissue, cause hypertension.^{35 36} Tobian and Redleaf³⁷ have proposed that aldosterone affects salt and water balance in vascular cells and thereby influences vessel lumen size. We have reported that vascular 11ß-HSDI and mRNA were decreased in DS rats.⁴ Franco-Saenz et al²⁹ also reported decreased 11ß-HSDI activity in the kidney of DS rats. Not only 11ß-HSDII but also 11ß-HSDI may play a role in salt sensitivity and development of hypertension in the DS rats. The decreased 11ß-HSDII activity in 8- or 12-week-old DS rats was not improved after treatment of hypertension with a calcium channel blocker (data not shown). This change in 11ß-HSDII activity in DS rats does not seem to be merely secondary to hypertension.

The excretion of endogenous 11ß-HSD inhibitory factor(s) has been reported in human urine.^{9 38} Glycyrrhetinic acid (GA), the active agent in licorice root, markedly inhibits 11ß-HSD when incubated with this enzyme. Morris et al³⁸ quantified this 11ß-HSD inhibitory factor(s) (glycyrrhetinic acid–like factors [GALFs]) using rat liver microsome and reported increased excretion in pregnancy. Walker et al³⁹ reported that concentrations of GALFs do not show diurnal rhythm and are unaffected by dexamethasone treatment in patients with low corticotropin or in patients with ectopic corticotropin secretion. They also reported that in hypertensive patients with impaired 11ß-HSD activity, GALF concentrations do not correlate with blood pressure, and they concluded that GALFs are unlikely to be involved in the pathophysiology of hypertension.⁴⁰ However, Semafuko et al⁴¹ demonstrated that urinary GALF was increased in patients with congestive heart failure. It was hypothesized that 11ß-HSDII inhibitory factors would serve to cause glucocorticoids, and possibly other steroids, to elicit Na⁺ retention by mineralocorticoid-mediated mechanisms and therefore augment, either naturally or in disease states, the Na⁺-retaining actions of aldosterone. We have reported that 11ß-HSDII inhibitory factors exist in human urine, and urinary excretion of these factors is increased in subgroups of hypertensive patients.⁹ Souness et al⁷ reported that 11- and 11ß-hydroxyprogesterone are potent inhibitors of 11ß-HSDII and are extremely active in conferring mineralocorticoid Na⁺-retaining activity on corticosterone in vivo in a rat bioassay. They also recently reported the hypertensinogenic activity of these progesterone metabolites in the rat.⁸ In this experiment, urinary excretion of 11ß-HSDII inhibitory factors was increased in DS rats compared with DR or SD rats. Lo et al¹⁰ reported that kidney 11ß-HSDII is inhibited by urinary 11ß-HSDII inhibitory factors extracted and partially purified from human urine. There is a possibility that increased 11ß-HSDII inhibitory factors may directly or indirectly decrease the 11ß-HSDII activity in kidney or blood vessel in hypertensive DS rats. Decreased 11ß-HSD mRNA levels might suggest that these factors operate at the transcriptional level. Further studies are needed to determine not only the chemical structures of the renal 11ß-HSDII inhibitory factor(s) but to reveal the source and their pathophysiological roles.

Received April 6, 1998; first decision April 27, 1998; accepted July 16, 1998.

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