Distal tubular electrolyte transport during inhibition of renal 11beta -hydroxysteroid dehydrogenase

Katharine J. Biller1,dagger, Robert J. Unwin2, and David G. Shirley2

1 Division of Biomedical Sciences, Imperial College School of Medicine, Charing Cross Hospital, London W6 8RF; and 2 Centre for Nephrology, Royal Free and University College Medical School, London W1N 8AA, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To test the proposal that the enzyme 11beta -hydroxysteroid dehydrogenase (11beta -HSD) confers aldosterone specificity on mineralocorticoid receptors in the distal nephron by inactivating glucocorticoids, we performed a free-flow micropuncture study of distal tubular function in adrenalectomized rats infused with high-dose corticosterone. One-half of the rats were additionally given intravenous carbenoxolone (CBX; 6 mg/h) to inhibit renal 11beta -HSD activity. Although this maneuver lowered fractional Na+ excretion (1.1 ± 0.2 vs. 1.9 ± 0.2%, P < 0.01), Na+ reabsorption within the accessible distal tubule was found to be similar in the two groups of animals. In contrast, distal tubular K+ secretion was enhanced in CBX-treated rats: fractional K+ deliveries to the early and late distal collection sites in the corticosterone-alone group were 13 ± 1 and 20 ± 3%, respectively (not significant), whereas corresponding data in the CBX-treated group were 9 ± 1 and 24 ± 2% (P < 0.01). This stimulation of distal K+ secretion provides the first direct in vivo evidence that 11beta -HSD normally prevents corticosterone from exerting a mineralocorticoid-like effect in the distal tubule. The reduction in fractional Na+ excretion during inhibition of 11beta -HSD, in the absence of a change in end-distal Na+ delivery, suggests enhanced Na+ reabsorption in the collecting ducts.

carbenoxolone; sodium reabsorption; potassium secretion; micropuncture; corticosterone


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN VITRO STUDIES HAVE DEMONSTRATED that mineralocorticoid receptors (MR) have equal affinity for aldosterone and glucocorticoids (26), yet in vivo they display specificity for aldosterone in the face of much higher circulating concentrations of glucocorticoids. It has been proposed that this specificity is conferred by the type 2 isoform of 11beta -hydroxysteroid dehydrogenase (11beta -HSD), an enzyme that converts glucocorticoids (cortisol in humans, corticosterone in rodents) to inactive 11-ketosteroid derivatives and is found in mineralocorticoid-sensitive tissues including the distal nephron (4, 8, 11).

This hypothesis has received considerable support from in vitro experiments (3, 11, 20) and from the observation that individuals with mutations of the gene encoding 11beta -HSD2 display the syndrome of apparent mineralocorticoid excess, a condition characterized by Na+ retention, hypertension, and hypokalemia in the absence of elevated aldosterone levels (33, 40); it has also been demonstrated that mice with targeted disruption of the 11beta -HSD2 gene are hypokalemic and hypertensive (16). To date, however, no direct in vivo confirmation of the proposed role for 11beta -HSD in the distal nephron has been provided.

11beta -HSD can be inhibited in vitro by glycyrrhetinic acid, which is the active constituent of licorice, or by its hemisuccinate derivative carbenoxolone (CBX) (8, 11, 20). In a recent clearance study, we infused CBX into intact, anesthetized rats and confirmed dose-dependent inhibition of renal 11beta -HSD, measured directly ex vivo (25). We were able to demonstrate a fall in Na+ excretion, in the absence of a change in glomerular filtration rate, when renal 11beta -HSD activity was inhibited by >90%. However, because clearance studies are unable to provide precise localization of tubular effects, the site of the enhanced Na+ reabsorption could not be identified. Furthermore, because the animals had intact adrenals, possible confounding influences of altered endogenous adrenocorticosteroid secretion could not be discounted.

In the present study, to test directly the proposed role of 11beta -HSD in the distal nephron, we have used in vivo micropuncture to investigate distal tubular function during CBX infusion in adrenalectomized, hormone-replaced rats. It was reasoned that, if the hypothesis were correct, inhibition of 11beta -HSD should prevent inactivation of infused glucocorticoid and thereby allow it to gain access to MR, leading to mineralocorticoid-like effects (increased Na+ reabsorption, increased K+ secretion) in the distal tubule.


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RESULTS
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Adrenalectomy

Adult male Sprague-Dawley rats weighing 220-240 g were adrenalectomized via bilateral flank incisions under halothane anesthesia (Fluothane, ICI, Macclesfield, Cheshire, UK). At the same time, a 14-day osmotic minipump (Alzet, model 2002, Alza, Palo Alto, CA) was implanted subcutaneously on the back of the neck of each animal. The pump contained a solution of D-aldosterone and dexamethasone (both from Sigma, Poole, Dorset, UK) dissolved in polyethylene glycol 400 (Sigma) so as to deliver basal levels of aldosterone (5 µg · kg body wt-1 · day-1) and dexamethasone (12 µg · kg body wt-1 · day-1) (10). All rats were given an analgesic injection of buprenorphine hydrochloride (Temgesic, 1.2 mg/kg sc; Reckitt & Colman, Hull, UK) and allowed to recover consciousness.

After adrenalectomy and minipump implantation, rats had free access to water and a standard rat diet containing 140 mmol Na+ and 180 mmol K+/kg dry wt. In addition, 2.7% (0.46 M) saline was provided as a drinking option up to the time of micropuncture experiments. Adrenalectomized animals drank 5.2 ± 0.5 (SE) ml/day of the hypertonic saline, whereas when it was offered as an option to adrenal-intact rats, negligible quantities were drunk. Rats were weighed immediately before surgery, 24 h after surgery, and then every day thereafter at the same time, to monitor weight gain.

Micropuncture Studies

Experiments were conducted 6-8 days postadrenalectomy, by which time the rats had gained 20-40 g compared with their preoperative weight. On the day of study, rats were anesthetized with Intraval (110 mg/kg ip, May & Baker, Dagenham, UK) and prepared surgically for micropuncture experiments (28). Rectal temperature was maintained at 37°C, a tracheotomy was performed, and the bladder was catheterized. The right femoral artery was cannulated for blood pressure monitoring and blood sampling. The left kidney was exposed by a subcostal incision, freed from perirenal fat, and immobilized in a Perspex dish rigidly attached to the operating table. The kidney surface was bathed with paraffin oil heated to 37°C. The left ureter was cannulated close to the pelvis. A total of four polyethylene intravenous cannulas were used: two were placed in the right jugular vein, one in the left jugular vein, and one in the right femoral vein. The left jugular vein cannula was used for Lissamine green injection, while the other three were for infusions as described below.

Control group (corticosterone alone; n = 12 rats). Corticosterone (Sigma), dissolved in isotonic saline containing 1.5% ethanol, was infused through one of the jugular cannulas (60 µg/ml, 4 ml/h) to administer corticosterone at 240 µg/h. Isotonic saline was infused at 1 ml/h through the right femoral cannula, making a total fluid infusion rate of 5 ml/h.

One hour after the end of surgery, a priming dose of 60 µCi of [3H]inulin (Amersham International, Aylesbury, UK) was given via the femoral vein cannula, followed by an infusion of 60 µCi [3H]inulin/h in isotonic saline (1 ml/h). This infusion replaced the initial saline infusion and was continued for the rest of the experiment.

CBX group (corticosterone and CBX; n = 12 rats). Corticosterone, dissolved in isotonic saline containing 2% ethanol, was infused through one of the jugular cannulas (80 µg/ml, 3 ml/h) to administer corticosterone at 240 µg/h. Isotonic saline was infused at 2 ml/h through the right femoral cannula.

One hour after the end of surgery, the saline infusion into the femoral vein was replaced by [3H]inulin in saline (60 µCi primer plus 60 µCi/h at 1 ml/h). At the same time, an infusion of CBX (Sigma; 6 mg/ml) was initiated through the second jugular cannula at a rate of 1 ml/h, making a total fluid infusion rate of 5 ml/h.

For both groups of rats, micropuncture collections were begun 1 h after the start of the [3H]inulin infusion. Throughout the micropuncture period (3-4 h), small arterial blood samples were taken for [3H]inulin measurements, and urine collections were made from both micropuncture and contralateral kidneys.

Samples of tubule fluid were collected from early and late distal puncture sites by using sharpened glass micropipettes (tip diameter 8-9 µm) filled with mineral oil containing Sudan black dye (39). Identification of distal tubules was made by using intravenous injections of Lissamine green dye (30 µl of 5% solution). These injections were separated by intervals of at least 45 min. For each collection, an oil column of about four to five tubular diameters in length was first injected into the tubule and allowed to flow to a position just downstream of the puncture site. Tubular fluid was then aspirated into the pipette (using initial gentle suction) at a rate that maintained the position of the oil column. At the end of each collection, silicone rubber solution (Microfil; Flow Tech, Carver, MA) was injected into the tubule to allow the subsequent confirmation of the collection site. Collection sites were accepted as "early" if they were in the first third of the distal tubule and as "late" if they were in the final third, the distal tubule being defined as that segment between the macula densa and the confluence with another distal tubule. Micropuncture collections were deposited under water-saturated oil, and their volumes were measured by using calibrated constriction pipettes. Duplicates of each sample were taken for measurement of [3H]inulin; triplicates of each sample were taken for tubular fluid Na+ and K+ measurement.

At the end of the experiment, small arterial blood samples were taken for measurement of pH and hematocrit, and a larger sample (~2 ml) was taken for measurement of plasma Na+, K+, osmolality, and (in 3 rats/group) corticosterone. The right kidney was removed, snap-frozen in liquid nitrogen, and stored for subsequent measurement of 11beta -HSD activity. The left kidney was removed and stored in distilled water at 4°C. It was subsequently partially digested in NaOH (5 M), and the silicone rubber-filled tubules were dissected out.

Assessment of the Effect of CBX in the Absence of Corticosterone

In view of a report that high-dose CBX might bind to MR and thus affect electrolyte excretion independently of an action on glucocorticoid metabolism (2), we assessed renal function in two further groups of adrenalectomized animals, prepared in exactly the same way as described above except that neither group received corticosterone. One hour after the end of surgery, both groups received intravenous [3H]inulin (2 µCi primer, 2 µCi/h). At the same time, CBX (6 mg/h) was infused in one group (n = 6 rats), whereas the other group (controls; n = 6 rats) received vehicle alone. Clearance measurements were made during the period 1-4 h after the start of CBX (or vehicle) infusion.

11beta -HSD Assay

11beta -HSD activities in homogenates of the snap-frozen kidneys removed at the end of the micropuncture studies were measured by using a standard radiometric conversion assay as previously described in detail (25). In essence, the conversion of [3H]cortisol (500 nmol/l) to [3H]cortisone in the presence of NAD+ or NADP+ was assessed by using thin-layer chromatography to quantify the product, and results were standardized according to the protein content of the homogenate.

Analyses

Arterial blood pressure was measured by using a Druck (Groby, Leicester, UK) pressure transducer. Urinary and plasma Na+ and K+ concentrations were measured by flame photometry (model 543, Instrumentation Laboratory, Warrington, UK). [3H]inulin activities in urine, plasma, and tubular fluid samples were determined by using beta -emission spectroscopy (model 2000 CA, Canberra Packard, Pangbourne, UK) after dispersal in Aquasol 2 scintillation cocktail (Canberra Packard). Arterial pH was measured with an ABL 500 blood-gas system (Radiometer, Copenhagen, Denmark); hematocrit was measured by using microhematocrit tubes (Hawksley & Sons, Lancing, UK); and plasma osmolality was measured by freezing-point depression (Roebling automatic osmometer, Camlab, Cambridge, UK). Tubular fluid Na+ and K+ concentrations were determined by using electrothermal atomic absorption spectrophotometry (Perkin Elmer atomic absorption spectrophotometer, model 3110, with HGA 600 furnace and AS 60 autosampler; Perkin Elmer, Beaconsfield, UK). Polypropylene volumetric flasks to be used for tubular fluid Na+ and K+ measurement were washed repeatedly with fresh 18 MOmega water (Elga UHQ water purification system, Elga, High Wycombe, UK) to avoid contamination. Fresh 18 MOmega water (140 µl) was then placed in each flask, and a volume of 9 nl of tubular fluid sample was added. The protocol for measurement of Na+ and K+ has been described in detail elsewhere (27).

Plasma aldosterone was measured by using radioimmunoassay [(14); Coat-A-Count Aldosterone kit, DPC, Los Angeles, CA]. Within-assay coefficients of variation (n = 15) were 6.9 (100-200 pmol/l) and 5.8% (200-400 pmol/l); between-assay coefficients of variation (n = 15) were 7.8 (100-200 pmol/l) and 6.9% (200-400 pmol/l). However, subsequent studies established that the antibody used in the assay cross-reacted with both corticosterone and CBX, thus invalidating the data. Accordingly, we measured plasma aldosterone in a separate group of adrenalectomized, hormone-replaced rats (n = 9) that had been prepared in the same way as the other rats in the present study except that neither corticosterone nor CBX was infused. The plasma aldosterone concentration of these rats was 188 ± 35 pmol/l.

Plasma corticosterone levels were also measured by radioimmunoassay (7). Within- assay and between-assay coefficients of variation were 9.2 (n = 10) and 9.4% (n = 10), respectively.

Calculations

Mean arterial blood pressure was calculated as diastolic pressure plus one-third of the pulse pressure. Glomerular filtration rate (GFR) was calculated as the renal clearance of [3H]inulin. The fractional excretions of Na+ (FENa), K+ (FEK), and water (FEH2O) were calculated as the clearances of Na+ and K+, and urine flow rate, respectively, divided by GFR. Single-nephron GFR (SNGFR) was calculated as TF/PIn multiplied by VTF, where TF/PIn is the tubular fluid/plasma concentration ratio for [3H]inulin and VTF is the tubular fluid flow rate. Fractional deliveries of Na+ and K+ to the distal collection sites were calculated as [(TF/PNa)/(TF/PIn)] × 100 and [(TF/PK)/(TF/PIn)] × 100, respectively, where TF/PNa and TF/PK are the tubular fluid/plasma concentration ratios for Na+ and K+.

Statistics

Results are presented as means ± SE. Statistical comparisons between the two groups of rats were made using Student's unpaired t-test. Within-group comparisons (i.e., early distal vs. late distal fractional deliveries) were assessed by using Student's paired t-test. A value of P < 0.05 was considered to be statistically significant.


    RESULTS
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INTRODUCTION
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Effect of CBX in the Presence of Corticosterone

Blood pressure and clearance data. Mean arterial pressure was not significantly different between the two groups of rats (103 ± 3 mmHg, corticosterone alone; 108 ± 4 mmHg, corticosterone+CBX). Overall clearance data for the left (micropunctured) kidney are shown in Fig. 1; values for the right kidney were very similar. Total GFR was similar in the two groups of animals. FEH2O was lower in the CBX-treated rats, as was FENa, but FEK was unaffected.


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Fig. 1.   Glomerular filtration rate (GFR) and the fractional excretion of water (FEH2O), sodium (FENa), and potassium (FEK) in the 2 groups of rats. Values are means ± SE for the left kidney only; n = 12 rats/group.

Blood and plasma data. Table 1 shows values from blood samples taken at the end of each experiment. Arterial pH, plasma osmolality, and plasma Na+ and K+ concentrations were all virtually identical in the two groups of rats. Although hematocrit tended to be lower in the CBX-treated animals (presumably as a consequence of reduced Na+ and water excretion in the face of a constant infusion rate), the apparent difference did not achieve significance.

                              
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Table 1.   Blood and plasma data in the 2 groups of rats

Micropuncture data. As with total GFR, there was no difference in SNGFR between the two groups of rats (37 ± 2 nl/min, corticosterone alone; 36 ± 2 nl/min, corticosterone+CBX). Values for tubular fluid flow rate, TF/PIn, TF/PNa, and TF/PK at the two puncture sites are shown in Table 2. Although tubular fluid flow rate in CBX-treated rats was, on average, slightly lower (and TF/PIn correspondingly higher) at each site, in neither case was there a significant difference between the two groups. The TF/P concentration ratio for Na+ was slightly, but not significantly, reduced at the early distal site in the CBX-treated rats; values at the late distal site were very similar in the two groups. However, for K+, the early distal TF/P concentration ratio was significantly lower in CBX-treated animals, whereas, in marked contrast, the late distal TF/PK was significantly elevated.

                              
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Table 2.   Tubular fluid flow rate and tubular fluid-to-plasma concentration ratios for [3H]inulin, Na+, and K+ in the 2 groups of rats

Figure 2 shows values for fractional Na+ delivery in the two groups of rats. Individual values, as well as group means, are presented. In every animal there was a marked reduction in fractional Na+ delivery between the early and late distal sites, but there was no discernible difference in Na+ handling between the groups: each group reabsorbed ~60% of the delivered Na+ load within the accessible distal tubule.


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Fig. 2.   Fractional Na+ deliveries at the early (ED) and late distal (LD) tubular sites in the 2 groups of rats. Open symbols connected by solid lines, individual values [i.e., average value for each site per rat; corticosterone (A) and corticosterone + CBX (B)]; closed symbols, mean (±SE) values (C). CBX, carbenoxolone.

Distal tubular K+ handling is shown in Fig. 3. In the corticosterone-alone group, although the mean fractional K+ delivery to the late distal site slightly exceeded that to the early distal site (Delta  = 7 ± 3%), the difference did not achieve statistical significance. In contrast, in every rat given CBX, fractional K+ delivery to the late distal site was greater than that to the early distal tubule; the mean difference (Delta  = 15 ± 3%) was therefore highly significant (P < 0.01) and exceeded that in corticosterone-alone rats (P < 0.05). Notably, the mean value for fractional K+ delivery at the early distal tubule in CBX-treated rats was significantly lower than that in the corticosterone-alone group (P < 0.01). At the late distal site, the mean value was slightly greater in the CBX-treated group, although at this site the apparent difference between the groups did not achieve statistical significance.


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Fig. 3.   Fractional K+ deliveries at the ED and LD tubular sites in the 2 groups of rats. Open symbols connected by solid lines, individual values [i.e., average value for each site per rat; corticosterone (A) and corticosterone + CBX (B)]; closed symbols, mean (±SE) values (C). The mean difference between fractional K+ deliveries to the ED and LD sites was significantly greater (P < 0.05) in CBX-treated rats than in corticosterone-alone rats. *P < 0.01 compared with corresponding value in the corticosterone group.

Enzyme assay. NAD+-dependent 11beta -HSD activity (pmol cortisol converted to cortisone per mg protein per 30 min), as measured ex vivo in the contralateral kidney, was 26 ± 5 pmol · mg-1 · 30 min-1 in the corticosterone-alone group and 6 ± 2 pmol · mg-1 · 30 min-1 in the corticosterone+CBX group. Corresponding values for NADP+-dependent 11beta -HSD activity were 49 ± 13 and 5 ± 1 pmol · mg-1 · 30 min-1. Thus CBX at 6 mg/h reduced enzyme activities to values similar to those seen previously with this dose (25).

Effect of CBX in the Absence of Corticosterone

Experiments designed to test the possibility that the dose of CBX used might itself have directly influenced renal electrolyte excretion, independently of an effect on corticosterone metabolism, yielded negative findings. In the control group (adrenalectomized, receiving no corticosterone infusion), FENa was 1.9 ± 0.5%; in the CBX-treated animals (adrenalectomized, receiving no corticosterone infusion, CBX infused at 6 mg/h), FENa was 2.3 ± 0.5%. Corresponding values for FEK were 33 ± 4 and 28 ± 2%, respectively. Thus no evidence for a direct mineralocorticoid-like effect was seen with this dose of CBX.


    DISCUSSION
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Two isoforms of 11beta -HSD exist within the rat kidney: type 1, found in the proximal tubule (1), the function of which is being examined elsewhere (6), and type 2, found in the distal nephron (1, 17, 29), which was the focus of the present investigation.

CBX has been used in a number of previous studies as a means of inhibiting 11beta -HSD to test the hypothesis that 11beta -HSD2 normally prevents access of glucocorticoids to MR and thus prevents Na+ retention. Although administration of CBX to humans and to rats was found to produce the anticipated reduction in Na+ excretion, in most studies no direct evidence that the enzyme had been inhibited was provided (18, 30, 34). In a recent investigation in this laboratory, we administered CBX to anesthetized rats and measured not only Na+ excretion but also renal 11beta -HSD activities, the latter assessed ex vivo by direct enzyme assay (25). We found that a dose of CBX sufficient to reduce 11beta -HSD activities by >90% caused a significant lowering of FENa (although smaller doses, which caused 50-80% enzyme inhibition, had no significant effect on excretion rates, suggesting either that 11beta -HSD has considerable functional reserve or that alternative mechanisms confer MR specificity). In that (clearance) study, it was not possible to ascertain the site of enhanced Na+ reabsorption. Moreover, adrenal-intact rats were used, and plasma aldosterone and corticosterone levels were therefore uncontrolled. Therefore, we performed the present micropuncture study in adrenalectomized rats.

The adrenalectomized, hormone-replaced rat model we employed was first developed by Martin et al. (19) and was subsequently used by Stanton and colleagues (10, 32) to assess the renal effects of aldosterone and glucocorticoids in the absence of anesthesia- and surgery-induced fluctuations in endogenous hormone levels. We found that plasma aldosterone concentrations, measured in preliminary studies in non-corticosterone-infused rats, were predictably low and similar to those reported previously in this model (32, 41). The fact that, before anesthesia, the rats drank significant quantities of hypertonic saline is further evidence that the replacement dose of aldosterone was not excessive, because rats will only drink saline of this strength if they are Na+ deficient (22, 23). Plasma corticosterone concentrations were measured in three rats from each group and ranged from 0.9 to 1.2 µmol/l; these are typical of values found in intact, stressed rats (C. Kenyon, personal communication). Thus sufficient corticosterone was present to test the hypothesis of a guardian role for 11beta -HSD.

The dose of CBX was the same as that previously shown to inhibit renal 11beta -HSD in intact rats by >90% (25). In the present study, as in the previous one, 11beta -HSD activities in CBX-treated rats were close to zero. The only difference between the two studies was that baseline values in adrenal-intact rats were greater than those in adrenalectomized animals. This finding, which conflicts with that of Alfaidy et al. (1), raises the possibility that enzyme activity is downregulated when adrenocortical hormones are chronically depleted. This would be consistent with recent evidence that 11beta -HSD activity is subject to physiological control (21, 36). Of the two isoforms of 11beta -HSD, type 1 can employ either NAD+ or NADP+ as a cofactor, whereas type 2 has an absolute requirement for NAD+. The fact that NAD+- and NADP+-dependent activities were both suppressed confirms that the CBX had inhibited both isoforms.

As a precaution, in view of an early report that very-high-dose CBX could bind to MR and thereby directly reduce the urinary Na+/K+ ratio (2), in the present study we administered CBX to a group of adrenalectomized rats that was not infused with corticosterone. Under these circumstances, no evidence for a reduction in Na+ excretion or in the urinary Na+/K+ ratio was seen. It is clear, therefore, that the CBX dose employed in our studies (6 mg/h) is insufficient to cause significant direct mineralocorticoid-like actions in vivo.

In contrast, in adrenalectomized rats infused with high-dose corticosterone, the effect of renal 11beta -HSD inhibition by this dose of CBX on overall electrolyte excretion was very similar to that previously observed in adrenal-intact animals: marked reductions in absolute Na+ excretion and FENa in the absence of a change in GFR, and no discernible effect on K+ excretion. The present micropuncture study, in which Na+ and K+ deliveries to the start and end of the distal tubule were measured directly, is the first investigation into the renal site(s) of action of CBX. At the early distal tubule, it was found that CBX treatment caused a significant reduction in K+ delivery when compared with values in rats infused with corticosterone alone. (The mean Na+ delivery was also slightly reduced, but the reduction did not achieve statistical significance.) Although there is some in vitro evidence that inhibition of proximal tubular 11beta -HSD may affect ion transport in that segment (6), the reduced early distal K+ delivery we observed was associated with a significant lowering of the TF/PK, which suggests increased K+ reabsorption in the loop of Henle rather than in the proximal tubule; any change in the latter would be expected to affect solute and water transport similarly. In this context, there is good evidence that aldosterone can stimulate K+ (and, to a much lesser extent, Na+) reabsorption in the thick ascending limb of Henle (TALH) (31). Because 11beta -HSD activity has been identified in microdissected segments of cortical TALH (4, 15, 17), it is tempting to speculate that CBX, by inhibiting 11beta -HSD in the TALH, was allowing corticosterone to exhibit mineralocorticoid-like activity in this nephron segment.

Leaving aside its effects in the loop, the major renal sites of action of aldosterone are generally believed to be the late distal tubule and cortical collecting tubule (24). However, autoradiographic studies have revealed aldosterone binding sites throughout the whole of the distal nephron, from distal convoluted tubule (DCT) to medullary collecting duct (9). The potential therefore exists for aldosterone to exert effects within the early, as well as late, distal tubule, a proposition supported by recent microperfusion evidence (37). In this context, although most studies of 11beta -HSD2 distribution have focused on cortical collecting tubule, it is becoming increasingly apparent that, like MR, the enzyme is present throughout the whole of the distal nephron (5, 15, 17, 38). If 11beta -HSD2 were to fulfill its putative guardian role in the distal nephron, it might therefore be expected that inhibition of the enzyme would allow access of corticosterone to MR in all aldosterone-sensitive segments (including the early distal tubule) and thereby stimulate Na+ reabsorption and K+ secretion. In the event, however, the present study was unable to provide any evidence for enhanced Na+ reabsorption in the accessible distal tubule of CBX-treated animals. (The distal tubule comprises the DCT, connecting tubule, and initial collecting tubule. It is likely that our "early distal" collection site was located within the DCT and our "late distal" site within the initial collecting tubule.) At first sight, this negative finding might suggest that the enzyme has no functional role to play in the distal nephron. However, it should be remembered that FENa was reduced in CBX-treated animals; moreover, K+ secretion in the accessible distal tubule was enhanced (see below). An explanation for the absence of an effect on distal tubular Na+ reabsorption may be found in the apparently conflicting findings of previous microperfusion studies of the action of aldosterone itself on distal tubular function. Stanton (31) reported that a chronic replacement dose of aldosterone in adrenalectomized rats restored Na+ reabsorption and K+ secretion to values indistinguishable from those in intact rats. Similarly, Velazquez and colleagues showed that chronic high-dose aldosterone could enhance distal tubular Na+ reabsorption in the adrenalectomized, hormone-replaced rat model (37). In contrast, the acute infusion of high-dose aldosterone into adrenalectomized, hormone-replaced rats, although producing the expected reduction in Na+ excretion, caused no enhancement of distal tubular Na+ reabsorption (but nevertheless stimulated K+ secretion) (10). Thus the time course of aldosterone's action on Na+ transport within the distal tubule may differ from that on K+ transport. In the present study, it is possible that the acute inhibition of 11beta -HSD is analogous to the acute infusion of aldosterone.

As already indicated, even though we found end-distal fractional Na+ deliveries to be identical in the two groups of rats, FENa was reduced in CBX-treated animals. One possible explanation for this could be that CBX somehow altered the relative end-distal Na+ deliveries from (accessible) superficial and (nonaccessible) juxtamedullary nephrons; i.e., that Na+ reabsorption in juxtamedullary nephrons was enhanced. (Unfortunately, no information is available on the distribution of 11beta -HSD between superficial and juxtamedullary nephrons.) The alternative, and in our view more likely, explanation for our findings is that inhibition of 11beta -HSD resulted in enhanced reabsorption of Na+ in the collecting duct.

In the absence of CBX, K+ delivery to the late distal tubule was not significantly greater than that to the early distal tubule. Thus the replacement dose of aldosterone employed was insufficient to stimulate net K+ secretion in the accessible distal tubule. In contrast, during inhibition of 11beta -HSD with CBX, there was clear evidence of K+ secretion: the fractional delivery of K+ to the late distal site was more than double that to the early distal tubule, and the late distal TF/PK was higher than the corresponding value in the corticosterone-alone group (in the absence of a difference in distal tubular water reabsorption between the 2 groups). The increase in distal tubular K+ secretion during CBX treatment occurred in the absence of an increase in tubular fluid flow rate (which was actually slightly depressed) or of alterations in arterial pH or plasma K+, factors known to influence K+ secretion (12). Stimulation of distal tubular K+ secretion after inhibition of 11beta -HSD is consistent with a mineralocorticoid-like effect of corticosterone, and, to our knowledge, provides the first in vivo evidence that inhibition of the enzyme leads to altered distal nephron function.

It may seem surprising, on the basis of present models of principal cell function, that the increase in distal tubular K+ secretion was unaccompanied by an increase in Na+ reabsorption. However, as noted above, there is a precedent for this finding in that acute aldosterone treatment causes a marked increase in distal tubular K+ secretion without a concomitant increase in Na+ reabsorption (10). Moreover, the magnitude of K+ secretion is small (in absolute terms) compared with Na+ reabsorption. In the present study, absolute Na+ reabsorption in the accessible distal tubule was ~300 pmol/min in both groups, whereas the difference in K+ secretion between the two groups amounted to ~10 pmol/min. Thus, even if the stoichiometry of Na+ and K+ transport in the distal tubule were assumed to be fixed, only a small percent change in Na+ reabsorption would be required to account for the change in K+ handling.

The effect of CBX on distal tubular K+ secretion was not accompanied by an increase in urinary K+ excretion, a situation that again mimics that seen during the acute infusion of high-dose aldosterone (10). It is unnecessary to infer from this that K+ must have been reabsorbed in the collecting duct, because the reduced fractional K+ delivery to the early distal tubule meant that, despite enhanced secretion, the fractional delivery to the late distal tubule was not significantly greater than that in rats given corticosterone alone. Nevertheless, if, as argued above, Na+ reabsorption in the collecting duct was increased during CBX treatment, it might be anticipated that K+ secretion would be enhanced in this nephron segment. That no such enhancement occurred could be attributable to a number of factors: 1) urine flow rate was lowered during CBX treatment, and a reduced flow rate through the cortical collecting duct would tend to offset any enhancement of K+ secretion; 2) not all the K+ secreted into the cortical collecting duct completes the journey to the final urine, as some is reabsorbed in the inner stripe of the outer medulla (35); and 3) in the in vitro situation, at least, aldosterone-stimulated Na+ reabsorption by rat inner medullary collecting duct cells occurs in the absence of altered K+ transport (13).

In conclusion, we suggest that the close similarity between the effects of 11beta -HSD inhibition, as documented in the present study, and the effects of intravenous infusion of high-dose aldosterone in the same rat model (10) provides further evidence that 11beta -HSD2 normally acts to protect MR in the distal nephron from the effects of circulating glucocorticoids.


    ACKNOWLEDGEMENTS

We thank the National Kidney Research Fund for financial support, J. Skinner and E. J. Folkerd for technical assistance, A. E. Michael and A. Thompson for enzyme assays, N. Payne for aldosterone measurements, and C. Kenyon for corticosterone measurements. We acknowledge the contribution of our valued colleague and trusted friend, the late Katharine J. Biller.


    FOOTNOTES

dagger Deceased.

Address for reprint requests and other correspondence: D. G. Shirley, Centre for Nephrology, Institute of Urology and Nephrology, Middlesex Hospital, Mortimer St., London W1N 8AA, UK.

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.

Received 24 September 1999; accepted in final form 31 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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