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Am J Physiol Regul Integr Comp Physiol 288: R1767-R1773, 2005. First published February 17, 2005; doi:10.1152/ajpregu.00786.2004
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RENAL HEMODYNAMICS AND CARDIORENAL INTEGRATION

Interactions between 11{beta}-hydroxysteroid dehydrogenase and COX-2 in kidney

Bing Yao,1 Raymond C. Harris,1 and Ming-Zhi Zhang1,2

George O'Brien Center for Kidney and Urologic Diseases and Departments of 1Cell and Developmental Biology and 2Medicine, Vanderbilt University School of Medicine and Department of Veterans Affairs Medical Center, Nashville, Tennessee

Submitted 19 November 2004 ; accepted in final form 7 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD2). In this disorder, cortisol is not inactivated by 11{beta}HSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11{beta}HSD2 inhibition and examined its possible role in the development of hypertension. 11{beta}HSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11{beta}HSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11{beta}HSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11{beta}HSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11{beta}HSD2 deficiency and that 11{beta}HSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.

prostaglandin synthase G2/H2; mineralocorticoid receptor; corticosterone; postnatal development

RENAL REGULATION of sodium and water excretion is central to the control of blood pressure and is mediated in large part by the adrenal mineralocorticoid hormone aldosterone (2). The receptor mediating aldosterone action, the mineralocorticoid receptor (MR), is relatively nonselective and can be equivalently activated by both glucocorticoid (GC) and mineralocorticoid (MC) (6, 15–17). Although plasma levels of GC are much higher than that of aldosterone, renal MRs are activated only by aldosterone under normal conditions. Specificity of MR renal action is achieved through inactivation of GCs by type 2 11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD2), which is highly expressed and colocalized with MR-positive epithelial cells in the kidney (6, 15–17). The syndrome of apparent mineralocorticoid excess (SAME) is caused by GC-induced activation of MR due to kidney 11{beta}HSD2 deficiency (13, 16). Reduced 11{beta}HSD2 activity also is found in other patients with increased salt sensitivity and blood pressure (19, 24).

Prostaglandins (PGs) are involved in regulating vascular tone and salt and water homeostasis in the mammalian kidney (8, 22). In the medulla, PGs act as diuretic and natriuretic agents by increasing blood flow in the vasa recta, decreasing salt reabsorption in the medullary thick ascending limbs of Henle's loop (mTAL), and reducing vasopressin-stimulated water reabsorption from collecting ducts (22). Cyclooxygenase (COX) is a rate-limiting step in PG production. Two isoforms of COX exist: COX-1 and COX-2 (8, 9). COX-2 is localized to the macula densa (MD) and surrounding cortical TAL in the cortex and to the interstitial cells in the medulla (7–9, 28–31). Renal medullary COX-2 is stimulated, and cortical COX-2 is suppressed, by MCs and high salt (HS) intake, whereas medullary and cortical COX-1 levels are relatively stable in response to these stimuli (7–9, 11, 26, 28–31). PGs derived from increased medullary COX-2 stimulate sodium and water excretion to counteract the increased salt retention. COX-2-selective and -nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to induce sodium retention (5, 12, 21, 25). NSAIDs primarily elevate preexisting hypertension (12). Selective COX-2 inhibitors also have been reported to elevate blood pressure in a minority of patients and to exacerbate existing hypertension (5, 8, 10, 21, 25, 27). The first aim of the current investigation was to examine whether 11{beta}HSD2 inhibition would stimulate medullary COX-2 expression through activation of MR and whether COX-2 inhibition would elevate blood pressure in normotensive and hypertensive rats.

Renal medullary COX-2 expression is related to urinary concentrating ability (30). Urinary concentrating ability is limited in suckling rats and matures after weaning. Circulating corticosterone levels are very low in neonates and reach adult levels after weaning (14, 31). Renal 11{beta}HSD2 expression is abundant in young and adult rats (14). The second aim of the present study was to compare medullary COX-2 expression in suckling and weaning rats and to investigate whether 11{beta}HSD2 plays a role in maintaining renal cortical and medullary COX-2 expression during postnatal development by adjusting GC-induced activation of MR.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The 11{beta}HSD2 inhibitor glycyrrhizic acid (GA) and the GC receptor (GR) antagonist RU486 were obtained from Sigma (St. Louis, MO). The MR antagonist eplerenone was obtained from Pfizer (New York, NY). The COX-1 inhibitor (SC-58560) and COX-2 inhibitor (SC-58236) were gifts from Searle Monsanto (St. Louis, MO).

Animals. Experiments conformed to ethical guidelines for animal research established by the Department of Animal Care in Vanderbilt University and received prior approval before the initiation of the experiments. Pregnant Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). Each litter was reduced to 8–10 pups just after birth. For studies of the effects on blood pressure in the adult rats (male, 225–250 g at the beginning of the experiments), GA, HS diet, SC-58236, and SC-58560 were administered for 3 wk. GA was given in the drinking water (0.1% GA) in adult and weaning rats or via subcutaneous injection of 1% GA solution dissolved in water at a dose of 0.1 g·kg–1·day–1 in weaning and suckling rats. HS was achieved by maintaining the rats with HS diet (8% NaCl; ICN Biochemicals, Costa Mesa, CA). SC-58236 and SC-58560 were given in the drinking water containing 0.2% polyethylene glycol (PEG200) and 0.01% Tween 20 (vol/vol), with 6 µg/ml SC-58236 or 60 µg/ml SC58560, respectively. RU486 (dissolved in sesame oil) was given at a dose of 7.5 mg·kg–1·day–1 by daily intramuscular injection for 7 days. Eplerenone (dissolved in water) was given at a dose of 0.1 g·kg–1·day–1 via gavage for 7 days. Control rats were fed with standard rodent diet containing 0.4% sodium (LabDiet 5001; PMI Nutrition International, Brentwood, MO). To determine plasma corticosterone and aldosterone levels, we treated all rats in the same way to collect blood samples. The animals were removed from the cage at 8:00 AM and immediately anesthetized with Nembutal at a dose of 70 mg/kg ip. Blood samples were collected from the heart, put into EDTA-coated tubes, and centrifuged. Plasma was collected and kept at –80°C until use. Plasma corticosterone or aldosterone levels were determined using RIA kits (ICN Biochemicals). To collect urine samples with maximal osmolality from rats at postnatal day 14 (P14), we removed the pup from the mother and placed it in a separate cage with one adult female rat. Because pups at P14 cannot empty their bladders without assistance from their mothers, at 15 h, when their bladders were easily palpable, they were emptied using a Credé maneuver. Three hours later, the pups were anesthetized with Nembutal (70 mg/kg ip), a midline abdominal incision was made, the bladder was visualized and punctured with a syringe, and urine was aspirated immediately from the bladder. To collect urine samples with maximal osmolality from weaning rats (P28), the bladder was emptied after the rat was water deprived for 33 h, and bladder urine was collected 3 h later.

Systolic blood pressure (SBP) was measured in conscious rats. Under anesthesia with Nembutal (60 mg/kg ip), a polyethylene catheter (PE-50) was inserted into the left common carotid artery. The catheter was tunneled under the skin, exteriorized, secured at the back of the neck, filled with heparinized saline, and sealed. The catheterized rat was housed individually in a metabolic cage and trained three times before measurement of blood pressure with a Blood Pressure Analyzer (Micro-Med, Louisville, KY).

Immunohistochemistry. In general, at the termination of an experiment, one kidney of each rat was removed for Western blot analysis and the other was perfused in situ for histology. Under deep anesthesia with Nembutal (70 mg/kg ip), each rat was exsanguinated with 50 ml/100 g heparinized saline (0.9% NaCl, 2 U/ml heparin, 0.02% sodium nitrite) through a transcardial aortic cannula and fixed with 3.7% formaldehyde in an acidic solution (pH 4.5) containing phosphate, periodate, acetate, and sodium chloride as described previously (9). The fixed kidney was dehydrated through a graded series of ethanols, embedded in paraffin, sectioned (4 µm), and mounted on glass slides. Internal controls and comparisons were facilitated by creating compound blocks with multiple specimens that were sectioned and stained together. COX-2 immunoreactivity (COX2-ir) was immunolocalized with affinity-purified rabbit polyclonal anti-murine COX-2 peptide (residues 570–598) antibody (no. 160126; Cayman Chemicals, Ann Arbor, MI) at a 0.1 µg IgG/ml dilution. The primary antibodies were localized by using Vectastain ABC-Elite (Vector, Burlingame, CA) with diaminobenzidine as chromogen, followed by a light counterstain with toluidine blue (9).

Immunoblotting. After being removed from the rat, the kidney was frozen in dry ice and stored at –80°C until use. Before analysis, the cortex and inner medulla (papilla) were dissected. Homogenates of cortex or papilla were prepared in 20 mM Tris-Cl, pH 8.0, with a proteinase inhibitor mixture (Boehringer Mannheim). After 10-min centrifugation at 10,000 g, the supernatant was centrifuged for 60 min at 100,000 g to sediment microsomes as described previously (9). A bicinchoninic acid (BCA) protein assay reagent kit was used to determine protein concentration. The microsomes were resuspended in homogenizing buffer, mixed with an equal volume of 2x SDS sample buffer, and boiled for 5 min. When Western blot analysis was performed, each lane was loaded with 50 µg (cortex) or 5 µg (papilla) of microsomes. The proteins were separated on 10% SDS-PAGE under reducing conditions and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). After blocking overnight with 20 mM Tris-Cl, pH 7.4, 500 mM NaCl, 5% nonfat milk, and 0.1% Tween 20, the blots were incubated for 3 h at room temperature with rabbit polyclonal anti-murine COX-2 at a 0.05 µg/ml dilution or goat polyclonal antihuman COX-1 at a 0.05 µg/ml dilution (Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibodies were detected with peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG and exposed on film by using enhanced chemiluminescence (Amersham International). Western blots were quantitated with an IS-1000 digital imaging system (Alpha Innotech, San Leandron, CA), the COX-2-ir band density from the control animal was designed as one, and that from the experiment animal was expressed as a relative multiple of control.

Micrography. Bright-field images from a Leitz Orthoplan microscope with a DVC digital RGB video camera were digitized using the BIOQUANT image analysis system and saved as computer files. Contrast and color level adjustment (Adobe Photoshop) were performed for the entire image; i.e., no region- or object-specific editing or enhancements were performed.

Statistical analysis. All values are presented as means ± SE. ANOVA and Bonferroni t-test were used for statistical analysis, and differences were considered significant when P < 0.05.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Effects of 11{beta}HSD2 inhibition on renal COX-2 and blood pressure in adult rats. The hypertension associated with either MC excess or SAME is sensitive to sodium intake (1). To investigate whether 11{beta}HSD2 inhibition mimics MC to stimulate medullary and suppress cortical COX-2 expression, we treated male adult rats with GA for 3 wk. HS was used as a positive control. As shown in Fig. 1, a significant decrease in cortical COX-2 (>10-fold decrease vs. control) and increase in medullary COX-2 (>4-fold increase vs. control) were observed in rats with either GA or HS treatment, similar to the effects of MC on renal COX-2 expression (28, 29). In contrast, neither cortical nor medullary COX-1 expression was affected by GA or HS treatment. Immunohistochemistry showed scattered COX-2-ir-positive cells in the cortex and abundant COX-2-ir-positive interstitial cells in the medulla in control rats (Fig. 2, A and C). In GA-treated rats, no apparent COX-2-ir-positive cells were found in the cortex, but the number of COX-2-ir-positive interstitial cells increased significantly in the medulla (Fig. 2, B and D). 11{beta}HSD2 inhibition resulted in significant decreases in plasma aldosterone levels (45 ± 7 vs. 145 ± 17 pg/ml for control, P < 0.01, n = 6), whereas plasma CS levels were unchanged [401 ± 52 vs. 425 ± 43 ng/ml in control, not significant (NS), n = 6].



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  		Fig. 1. Renal cortical cyclooxygenase-2 (COX-2) decreased (A) and medullary COX-2 increased (B) after treatment with either glycyrrhizic acid (GA) or high-salt diet (HS) for 3 wk. Neither cortical nor medullary COX-1 expression was affected by GA treatment. Insets: representative blots. C, cortical; M, medullary, ir, immunoreactivity. Values are means ± SE; n = 6. *P < 0.01 vs. control.

 


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  		Fig. 2. Renal COX-2 immunostaining in adult (A–D) and suckling rats (E–H). In adult rats, cortical COX-2 expression was lower after GA treatment (B) than in control rat (A), whereas medullary COX-2 expression was higher after GA treatment (D) than in control rat (C). In normal suckling rats, cortical COX-2 expression (E) was much higher and was not affected by GA treatment (F). Only scattered COX-2-ir-positive cells were found in medullary interstitium in normal suckling rat (G). GA treatment had no effect on medullary COX-2 expression in suckling rat (H). Relative width: 48 µm in A–D, G, H; 75 µm in E and F.

 
SBP was measured in conscious rats by using chronic indwelling catheters (Fig. 3). The rats were divided into eight groups: normal control, GA, HS, HS+SC-58236, HS+SC-58560, GA+HS, GA+HS+SC-58236, and GA+HS+SC-58560. All treatments lasted for 3 wk. HS alone had no effect on SBP (132 ± 6 vs. 127 ± 8 mmHg for control, NS, n = 6). GA increased SBP in rats with HS intake but not in rats with normal salt intake (control: 127 ± 8 mmHg, n = 6; GA: 125 ± 4 mmHg, NS vs. control, n = 6; GA+HS: 158 ± 5, P < 0.01 vs. GA, n = 4), indicating that GA induces salt-sensitive hypertension.



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  		Fig. 3. Effects of different treatments on systemic blood pressure (SBP) measured by common carotid artery in conscious rats. HS alone had no effect on blood pressure. GA increased blood pressure in HS-treated rats but not in normal salt-treated rats. Neither COX-1 (SC-58560) nor COX-2 inhibition (SC-58236) had an effect on blood pressure in HS-treated rats. COX-2 inhibition increased blood pressure in GA- and HS-treated rats; COX-1 inhibition did not. Values are means ± SE; n = 4–6.*P < 0.01 vs. control group. {dagger}P < 0.01 vs. GA+HS.

 
We hypothesized that in GA-treated rats, PGs derived from increased medullary COX-2 stimulate sodium and water excretion to counteract the increased salt retention, and COX-2 inhibition would be expected to result in more sodium retention and possibly subsequent elevation of blood pressure. The effects of COX-2 or COX-1 inhibition on blood pressure were investigated in normotensive (HS alone) and hypertensive (GA+HS) rats. In normotensive rats, administration of either the COX-2 inhibitor SC-58236 or the COX-1 inhibitor SC-58360 throughout the experiments did not affect SBP (HS: 132 ± 6 mmHg, n = 6; HS+SC-58236: 130 ± 5, NS vs. HS, n = 4; HS+SC-58360: 129 ± 6, NS vs. HS, n = 4). However, in hypertensive rats, administration of the COX-2 inhibitor SC-58236 throughout the experiment significantly increased SBP, whereas the COX-1 inhibitor did not (GA+HS: 158 ± 5, n = 4; GA+HS+SC-58236: 182 ± 8, P < 0.01 vs. GA+HS, n = 6; GA+HS+SC-58360: 152 ± 8, NS vs. GA+HS, n = 6). These results indicate that elevated renal medullary COX-2 due to 11{beta}HSD2 inhibition may attenuate the development of hypertension by facilitating sodium and water excretion.

Renal medullary COX-2 expression in young rats. In adult rats, renal medullary COX-2 expression decreases in response to salt restriction and impaired urinary concentrating ability (7, 11, 26, 30). During postnatal development of the rat, the neonate is in relative volume depletion and has limited urinary concentrating ability (31). After weaning, the urinary concentrating ability matures. Medullary COX-2 expression was compared between normal suckling (P14) and weaning rats (P28). As shown in Fig. 4A, medullary COX-2 expression was significantly lower in suckling than in weaning rats, consistent with low maximal urine osmolality in suckling rats and high maximal urine osmolality in weaning rats (P14: 315 ± 53 vs. P28: 1,166 ± 172 mosmol/kgH2O, P < 0.01, n = 8). Immunohistochemistry showed that there were much fewer interstitial cells in the papilla of suckling rat, and only a few COX-2-ir-positive interstitial cells were found (Fig. 2G).



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  		Fig. 4. Renal medullary COX-2 expression (A) and urinary concentrating ability (B) in suckling and weaning rats. A: renal medullary COX-2 expression was significantly lower in rats at postnatal day 14 (P14) than at P28. Inset: representative blot. B: maximal urine osmolality was lower in rats at P14 than at P28. Values are means ± SE; n = 8. *P < 0.01 vs. P14.

 
Effects of 11{beta}HSD2 inhibition on renal COX-2 expression in young rats. During postnatal development, plasma corticosterone levels are very low in neonates and subsequently increase to adult levels by weaning (14, 31). Although 11{beta}HSD2 expression is higher in young than in adult rats, it is expected to regulate renal COX-2 expression in weaning as well as adult rats but not in suckling rats because of low plasma corticosterone levels in these animals (14). Preliminary data showed that administration of GA via either subcutaneous injection or drinking water for a week had similar effects on renal COX-2 expression in weaning rats. In weaning rats, activation of MR in response to GA treatment was indicated by decreased urinary Na+/K+ ratios during the first 3 days of GA administration in the drinking water (urinary Na+/K+: day 0, 0.68 ± 0.07, day 3: 0.49 ± 0.05, P < 0.05 vs. day 0, n = 8). As shown in Fig. 5A, 11{beta}HSD2 inhibition (from P21 to P28) led to medullary COX-2 stimulation and cortical COX-2 suppression in weaning rats. Cortical COX-2 suppression was significantly attenuated by RU486 and completely reversed by eplerenone, whereas medullary COX-2 stimulation was slightly attenuated by RU486 and completely reversed by eplerenone. 11{beta}HSD2 inhibition also led to decreased plasma aldosterone levels in weaning rats (52 ± 15 vs. 161 ± 43 pg/ml for control, P < 0.01, NS, n = 8). As expected, 11{beta}HSD2 inhibition (from P7 to P14) had no effect on either cortical or medullary COX-2 expression (Figs. 2H and 5B) or plasma aldosterone levels (96 ± 27 vs. 84 ± 21 pg/ml for control, NS, n = 8).



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  		Fig. 5. Effects of 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}HSD2) inhibition with GA for 7 days on renal COX-2 expression in young rats. A: in weaning rats, GA treatment led to cortical COX-2 suppression and medullary COX-2 stimulation, which were attenuated by the glucocorticoid receptor antagonist RU486 but completely reversed by the mineralocorticoid receptor antagonist eplerenone. B: 11{beta}HSD2 inhibition had no effect on renal cortical or medullary COX-2 expression in suckling rats. Insets: representative blots. Values are means ± SE; n = 8. *P < 0.01 vs. control group. {dagger}P < 0.01 vs. GA.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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The major findings of these studies are 1) 11{beta}HSD2 inhibition induces salt-sensitive hypertension when COX-2 is inhibited; 2) medullary COX-2 expression is low in suckling rats, consistent with limited urinary concentrating ability; and 3) 11{beta}HSD2 inhibition leads to medullary COX-2 stimulation and cortical COX-2 suppression in weaning as well as adult rats but not in suckling rats, primarily by activation of MR, suggesting that 11{beta}HSD2 is involved in the regulation of renal COX-2 expression during postnatal development, primarily by limiting GC-induced activation of MR.

In mammalian kidney, GC and MC bind to MR with equal affinity (6, 15–17). MR, GR, and 11{beta}HSD2 are all expressed in distal nephron (3, 4, 20, 23). Renal cortical COX-2 expression is tonically suppressed by activation of MR and GR, whereas renal medullary COX-2 expression is stimulated primarily by activation of MR (28, 29). In weaning and adult rats, 11{beta}HSD2 inhibition increases intracellular corticosterone levels, leading to activation of MR and GR and subsequent cortical COX-2 suppression and medullary COX-2 stimulation. As shown in Figs. 2 and 5, 11{beta}HSD2 inhibition suppresses cortical and stimulates medullary COX-2 expression in weaning and adult rats, accompanied by decreased circulating aldosterone concentrations and decreased ratios of urinary Na+/K+. COX-1 expression is resistant to 11{beta}HSD2 inhibition. Cortical COX-2 suppression due to 11{beta}HSD2 inhibition is significantly attenuated by the GR antagonist RU486 but is completely reversed by the MR antagonist eplerenone (Fig. 5A). In contrast, medullary COX-2 elevation due to 11{beta}HSD2 inhibition is only slightly attenuated by RU486 but is completely reversed by eplerenone (Fig. 5A). These results indicate that the observed decrease in cortical COX-2 and increase in medullary COX-2 following 11{beta}HSD2 inhibition was primarily caused by activation of MR. In the cortex, 11{beta}HSD2 is primarily expressed in the distal convoluted tubule, connecting tubule, and cortical collecting tubule. 11{beta}HSD2 expression has been reported to be low in cortical TAL and MD, in which COX-2 is expressed in the cortex (20). 11{beta}HSD2 inhibition is expected to result in activation of MR primarily in the distal convoluted tubule, connecting tubule, and cortical collecting tubule but not in cortical TAL and MD. Therefore, cortical COX-2 decrease due to 11{beta}HSD2 inhibition is most unlikely caused by direct suppression via activation of MR but by indirect suppression via activation of MR secondary to positive sodium balance. In the medulla, COX-2 is primarily expressed in the interstitial cells in the inner medulla/papilla, in which 11{beta}HSD2 expression is undetectable or very low. In cultured mouse renal medullary interstitial cells, DOCA has no effect on COX-2 expression. Therefore, medullary COX-2 increase due to 11{beta}HSD2 inhibition is also likely an indirect response to activation of MR secondary to positive sodium balance (7, 26, 28).

In the medulla, PGs act as diuretic and natriuretic agents to promote sodium and water excretion (8, 22). Medullary COX-2 inhibition is expected to result in excessive sodium and water retention and elevation of blood pressure. As demonstrated in Fig. 3, either COX-1 or COX-2 inhibition alone has no effect on blood pressure in normotensive (HS alone) rats. However, COX-2 inhibition, but not COX-1 inhibition, augments elevated blood pressure in GA- and HS-treated rats. Our results are in agreement with clinical data showing that specific COX-2 inhibitors or other NSAIDs primarily elevate preexisting hypertension (12). COX-2 is also suggested as a major source of vascular endothelial PGI2, a potent vasodilator (10). The hypertensive effect of COX-2 blockade in our studies is therefore likely attributable to inhibition of COX-2-derived prostanoids that mediate the renal tubular and vascular response to excess activation of MR. It is somewhat surprising that GA treatment alone did not increase blood pressure, which may be attributable to the relatively short period of treatment.

Sodium retention, edema, and hypertension are often observed in patients treated with NSAIDs, and increased blood pressure after treatment with NSAIDs is observed primarily in patients with preexisting hypertension (5). In anesthetized mice, COX-2 inhibition decreased medullary blood flow as well as urinary volume and sodium excretion, whereas COX-1 inhibition had no effect on medullary hemodynamics (18), suggesting the importance of COX-2 in regulating renal sodium and water excretion. Recently, Zewde and Mattson (27) reported that chronic infusion into rat kidney medullary interstitial space of the COX-2 inhibitor NS-398 or an antisense oligonucleotide for COX-2 led to salt-sensitive hypertension, whereas intravenous infusion of NS-398 did not raise blood pressure, indicating local medullary rather than systemic effects. In their studies, similar concentrations of NS-398 (10 mg·kg–1·day–1) were administered intravenously or direct infusion into the medullary interstitial space. It is likely that the relative effectiveness of direct medullary infusion was due to the ability to achieve relatively high levels of NS-398 in the interstitial space.

In the current studies, the lack of response of suckling rats to 11{beta}HSD2 inhibition correlated with low circulating glucocorticoid levels. In addition, suckling rats are always in a state of relative volume depletion because sodium supply through maternal milk is highly limited (31). Excess activation of MR due to 11{beta}HSD2 inhibition in these rats would not be expected to result in further increase in sodium reabsorption and subsequent alterations of renal COX-2 expression. Therefore, relative volume depletion may be primarily responsible for the lack of effect of 11{beta}HSD2 inhibition on renal COX-2 in the suckling rats. This is supported by our previous report (28) that COX-2 decreases in the cortex and increases in the medulla due to DOCA administration are significantly attenuated in adult rats fed a low-salt diet. Therefore, in addition to low circulating levels of corticosterone, relative volume depletion may also contribute to the failure of suckling rats to alter renal COX-2 expression in response to 11{beta}HSD2 inhibition.

In adult rats, renal medullary COX-2 expression is related to kidney concentrating ability. In Brattleboro rats, kidney concentrating ability is impaired because of central vasopressin deficiency, and renal medullary COX-2 expression is very lower compared with fellow Long-Evans rats (30). Vasopressin supplementation increases both urine concentrating ability and medullary COX-2 expression (30). During postnatal development, urinary concentrating ability is limited in neonates and matures after weaning. In the present study, we found that renal medullary COX-2 expression in suckling rat at P14 is significantly lower compared with that in the weaning rat at P28. Whether limited urinary concentrating ability in neonates contributes to low levels of medullary COX-2 needs to be further investigated.

GA is the major bioactive triterpene glycoside of licorice root extracts. Because GA has a wide range of pharmacological properties, it remains possible that it may also affect COX-2 expression through MR-independent pathways. Because GA is not a specific inhibitor of 11{beta}HSD2, selective disruption of 11{beta}HSD2 gene in the distal tubule would provide an ideal model in which to study the role of GA in regulation of renal COX-2 expression.

In summary, renal 11{beta}HSD2 plays a role in maintaining renal cortical and medullary COX-2 expression during postnatal development by adjusting GC-induced activation of MR. Increased medullary COX-2 expression in response to 11{beta}HSD2 inhibition may serve to modulate the development of hypertension in this condition, and COX-2 inhibition may exacerbate the preexisting hypertension.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the Vanderbilt George O'Brien Kidney and Urologic Diseases Center [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-39261], NIDDK Grant DK-62794, and by funds from the Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Harris, C-3121 Medical Center North, Dept. of Medicine, Vanderbilt Univ., Nashville, TN 37232-4794 (E-mail: ray.harris{at}vanderbilt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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