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Published ahead of print on August 17, 2005
J Am Soc Nephrol 16: 2864-2871, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2004110944
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Cell and Transport Physiology

Molecular Mechanisms of Impaired Urinary Concentrating Ability in Glucocorticoid-Deficient Rats

Yung-Chang Chen*,{dagger}, Melissa A. Cadnapaphornchai*,{ddagger}, Sandra N. Summer*, Sandor Falk*, Chunling Li*, Weidong Wang* and Robert W. Schrier*

* Departments of Medicine and {ddagger} Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado; and {dagger} Division of Critical Care Nephrology, Chang Gung Memorial Hospital, Taipei, Taiwan

Address correspondence to: Dr. Robert W. Schrier, Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box B173, Denver, CO 80262. Phone: 303-315-8059; Fax: 303-315-2685; E-mail: robert.schrier{at}uchsc.edu

Received for publication November 16, 2004. Accepted for publication July 13, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of this study was to examine urinary concentrating ability and protein expression of renal aquaporins and ion transporters in glucocorticoid-deficient (GD) rats in response to water deprivation as compared with control rats. Rats underwent bilateral adrenalectomies, followed only by aldosterone replacement (GD) or both aldosterone and dexamethasone replacement (control). As compared with control rats, the GD rats demonstrated a decrease in cardiac output and mean arterial pressure. In response to 36-h water deprivation, GD rats demonstrated significantly greater urine flow rate and decreased urine osmolality as compared with control rats at comparable serum osmolality and plasma vasopressin concentrations. The initiator of the countercurrent concentrating mechanism, the sodium-potassium-2 chloride co-transporter, was significantly decreased, as was the medullary osmolality in the GD rats versus control rats. There was also a decrease in inner medulla aquaporin-2 (AQP2) and urea transporter A1 (UT-A1) in GD rats as compared with control rats. There was a decrease in outer medulla Gs{alpha} protein, an important factor in vasopressin-mediated regulation of AQP2. Immunohistochemistry studies confirmed the decreased expression of AQP2 and UT-A1 in kidneys of GD rats as compared with control. In summary, impairment in the urinary concentrating mechanism was documented in GD rats in association with impaired countercurrent multiplication, diminished osmotic equilibration via AQP2, and diminished urea equilibration via UT-A1. These events occurred primarily in the relatively oxygen-deficient medulla and may have been initiated, at least in part, by the decrease in mean arterial pressure and thus renal perfusion pressure in this area of the kidney.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability to conserve water during periods of fluid deprivation is an important function of the kidney. In both humans and experimental animals, adrenal insufficiency has been associated with several alterations in renal function, including impairment of urinary diluting and concentrating capacity (1–7). Isolated glucocorticoid deficiency has also been associated with impaired urinary concentration (8,9). However, the mechanisms of this defect at the cellular and molecular levels have not been defined.

In this study, glucocorticoid-deficient (GD) rats were compared with glucocorticoid-replete rats with respect to their capacities to concentrate the urine. Critical components of urinary concentration in response to fluid deprivation include release of the antidiuretic hormone arginine vasopressin (AVP) and upregulation of the abundance of aquaporin-2 (AQP2) water channels in the principal cells of the collecting duct. Moreover, activation of the countercurrent concentrating mechanism, which is initiated by the sodium-potassium-2 chloride (Na-K-2Cl) co-transporter in the water-impermeable ascending limb, creates the osmotic driving force for passive water reabsorption across the collecting duct. There are also roles for other water channels, including aquaporins 1, 3, and 4, and for urea transporters in urinary concentration. This study was undertaken to define the effect of glucocorticoid deficiency on these various molecular events during fluid deprivation in the rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Model
The study protocol was approved by the University of Colorado Institutional Animal Care and Use Committee. Male Sprague-Dawley rats that weighed 175 to 200 g were allowed to acclimate to Denver’s altitude (1500 m) for 1 wk before any experimental protocols. All animals underwent acclimation to metabolic cages for a continuous 5-d period before initiation of study. The animals were housed individually in metabolic cages and exposed to a 12-h light-dark cycle and constant ambient temperature. Eighteen rats were divided equally into each of two study groups: GD and control. Under anesthesia with ketamine (40 mg/kg body wt intraperitoneally) and xylazine (5 mg/kg body wt intraperitoneally), all animals were adrenalectomized through bilateral flank incisions. Simultaneously, osmotic minipumps (Alzet Osmotic Pump model 2ML4; Durect, Cupertino, CA) that contained aldosterone (Research Plus, Bayonne, NJ) at a dose calculated to deliver 17 µg/kg per 24 h into the peritoneal cavity were implanted into GD rats (10). Control rats received combined treatment with osmotic minipumps that contained aldosterone plus subcutaneous injections of dexamethasone (Research Plus) dissolved in peanut oil at a dose of 12 µg/kg per d starting immediately after adrenalectomy. This dose of dexamethasone has been reported to maintain normal weight gain, GFR, and fasting plasma glucose and insulin levels in adrenalectomized rats (11). For this study, we elected to use hormone-replaced adrenalectomized rats as controls, rather than intact (unaltered) animals. Our studies (data not shown) demonstrated that the protein abundance of inner medulla urea transporter A1 (UT-A1) was similarly diminished in the intact rats and in our controls.

After adrenalectomies, all rats were pair-fed with plain powdered rat chow (Harlan Teklad Bioproducts, Indianapolis, IN) 15 g/d. Drinking water was provided ad libitum. All animals were maintained in metabolic cages for the duration of the study to assess accurately daily food intake, water intake, and urine output.

On day 7 after adrenalectomies, echocardiography was performed using a GE Vingmed System 5 imaging tool (GE, Horten, Norway) for small rodents with a 10-MHz probe. The animals were anesthetized for echocardiography with ketamine and xylazine in doses as described above. Cardiac output was calculated via measurement of the diameter of the left ventricular outflow tract (LVOT), the flow through the outflow tract (VTI), and the heart rate (HR) by the formula, 0.785 x LVOT2 x VTI x HR. The right femoral artery then was catheterized with a polyethylene tube (PE-50; Intramedic, Clays Adams, Parsippany, NJ), and BP was measured using a Transpac disposable transducer (Abbott Critical Care Systems, Salt Lake City, UT) connected to a Transonic Systems T106 BP monitor (Ithaca, NY). BP was analyzed using WinDaq software (Dataq Instruments, Akron, OH). Cardiac output was factored by body weight and expressed as cardiac index (CI; ml/min per 100 g). Total peripheral resistance (mmHg/min per ml/100 g) was calculated by dividing mean arterial pressure (MAP) by CI. Stroke volume (ml/beat per 100 g) was obtained by dividing CI by HR (12,13). The catheter was removed. Animals were allowed to recover and then were returned to metabolic cages.

Two days after echocardiography, all animals were subjected to a 36-h period of water deprivation, during which time food intake and urine output were recorded. Urine was collected under oil, and urine volume was measured every 12 h. In the final 12 h of the water deprivation period, urine was collected for osmolality and creatinine and urea concentrations. Animals then were killed by decapitation to avoid any influence of anesthesia on plasma AVP concentration (14). Trunk blood was collected for plasma AVP concentration, blood urea nitrogen, serum glucose, serum osmolality, serum sodium, and serum creatinine concentration.

Protein Isolation
After decapitation, kidneys were placed in ice-cold isolation solution that contained 250 mM sucrose, 25 mM imidazole, 1 mM EDTA (pH 7.2), with 0.1% vol protease inhibitors (0.7 µg/ml pepstatin, 0.5 µg/ml leupeptin, and 1 µg/ml aprotinin), and 200 µM PMSF. Kidneys were dissected on ice into cortex, outer medulla, and inner medulla regions. Tissue samples were homogenized immediately in a glass homogenizer at 4°C. After homogenization, protein concentration was determined for each sample by the Bradford method (Bio-Rad, Richmond, CA). Tissue protein was used for immunoblotting for AQP water channels and sodium and urea transporters.

Western Blot Analysis
Western blot analysis was performed to examine expression of AQP1, AQP2, and Gs{alpha} subunit proteins in the renal cortex, outer medulla, and inner medulla; the Na-K-2Cl co-transporter, sodium-potassium-adenosine triphosphatase (Na-K-ATPase) {alpha}1 and {beta}1 subunits, and sodium-hydrogen exchanger (NHE3) in the renal cortex and outer medulla; AQP3 and UT-A2 in the outer medulla and inner medulla; and UT-A1 and AQP4 in the inner medulla. SDS-PAGE was performed on 8% acrylamide gels for the Na-K-2Cl co-transporter, Na-K-ATPase {alpha}1 subunit, and UTA and on 12% acrylamide gels for AQP, NHE3, Na-K-ATPase {beta}1 subunit, and Gs{alpha} subunit proteins. After transfer by electroelution to polyvinylidene difluoride membrane (Millipore, Bedford, MA), blots were blocked overnight with 5% nonfat dried milk in PBS(–) and then probed with the respective antibodies for 24 h at 4°C. After washing with buffer that containing PBS(–) with 0.1% Tween 20 (J.T. Baker, Phillipsburg, NJ), the membranes were exposed to secondary antibody for 1 h at room temperature. Subsequent detection of the specific proteins was carried out by enhanced chemiluminescence (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. Prestained protein markers were used for molecular mass determinations. Densitometric results were reported as integrated values (area x density of band) and expressed as a percentage compared with the mean value in controls (100%). Membranes were stained with Coomassie blue to ensure equal loading. For each gel, an identical gel was run in parallel and subjected to Coomassie staining to verify identical protein loading. Blots shown in the Results section are representative of the results obtained from all samples. Densitometry as shown in the Results section reflects means ± SEM densitometry of all 18 samples.

Antibodies
Antibodies to AQP2, AQP3, AQP4, NHE3, UT-A1, and UT-A2 have been characterized previously (15–19). Anti–Na-K-ATPase {alpha}1 and {beta}1 antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Antibodies to AQP1 and the Na-K-2Cl co-transporter were obtained from Chemicon International, Inc. (Temecula, CA). Anti-Gs{alpha} antibody was obtained from Calbiochem-Novabiochem (San Diego, CA).

Immunohistochemical Studies
Tissue samples were fixed in 4% formaldehyde solution for 24 h, dehydrated, embedded in paraffin, and cut into 2-µm-thick slices. Immunohistochemical staining was performed using the avidin-biotinylated peroxidase method. After deparaffinization and rehydration, sections were pretreated with 0.3% hydrogen peroxidase in 70% methanol to exhaust endogenous peroxidase activities. Sections were preincubated with 10% horse serum, then incubated with the antibodies to UT-A at a 1:50 dilution and AQP2 at a 1:200 dilution at 37°C for 1 h. Slides were washed and incubated with biotinylated secondary antibody, goat anti-rabbit IgG, at a 1:400 dilution in PBS. After treatment of the slides with an Elite ABC kit (Vector Laboratories, Burlingame, CA), antigens were visualized with the Sigma (St. Louis, MO) fast 3,3-diaminobenzidine tablet system applied on the slides for 1 min (AQP2) and 5 min (UT-A). Counterstaining was performed with Mayers’ hematoxylin (Fluka, Buchs, Switzerland) for 30 s.

Biochemical Measurements
Plasma AVP concentration was assessed by RIA as described previously (14). Serum and urine osmolality was measured by freezing point depression (Advanced Instruments, Inc., Norwood, MA). Serum and urine urea nitrogen and creatinine were measured (Beckman Instruments, Inc., Fullerton, CA). Twenty-four-hour creatinine clearance was used as an estimate of GFR. Serum sodium concentrations were measured by flame photometry. Blood glucose level was measured using a single-touch glucometer.

Medullary Tonicity
Samples of inner medulla were placed in a preweighed Eppendorf tube that contained 200 µl of deionized distilled water. The tissue was homogenized in a glass homogenizer at 4°C. Tissue osmolality was measured by freezing point depression (Advanced Instruments, Inc.). The original tissue osmolality was estimated on the basis of the nominal dilution factor and the assumption that 80% of the wet weight was water (20).

Statistical Analyses
Statistical analysis of results was performed using the unpaired t test. Results were expressed as means ± SEM with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systemic Hemodynamics in GD and Control Rats
Systemic hemodynamic studies revealed significantly decreased MAP and cardiac index in GD rats (n = 9) as compared with control rats (n = 9; Table 1). Neither calculated total peripheral resistance nor HR increased in response to the cardiac output–mediated decrease in MAP.


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  		Table 1. Systemic hemodynamic data obtained on day 7 after adrenalectomies in GD and control ratsa

 
Water Deprivation Studies
All rats then were subjected to a 36-h period of water deprivation. During this period, GD rats demonstrated significantly higher urine flow rate (Figure 1A) than control rats. In addition, maximal urine osmolality was significantly decreased in GD rats in response to water deprivation as compared with control rats (Figure 1B). The medullary osmolality was decreased to a similar degree as urinary osmolality in the GD rats (Figure 1C). After the 36-h water deprivation period, GD rats demonstrated a significantly lower serum glucose concentration than control rats. Fractional excretion of urea was significantly increased in GD rats after water deprivation as compared with control rats. Creatinine clearance, serum creatinine, sodium, and osmolality were similar between the study groups. No significant differences in plasma AVP concentrations were found in response to water deprivation. These results are shown in Table 2.



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  		Figure 1. Glucocorticoid deficient (GD) rats demonstrated increased urine flow rate (A), decreased maximal urinary osmolality (B), and decreased medullary osmolality (C) in response to 36-h water deprivation as compared with control (CTL) rats.

 

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  		Table 2. Characteristics of study groups after 36-h period of water deprivationa

 
Alterations to Renal Cortex, Outer Medulla, and Inner Medulla with Fluid Deprivation
Immunoblotting studies were performed for ion transporters and AQP water channels as described below for both GD (n = 7) and control (n = 8) rats. Blots shown are representative of all samples. Densitometric results were reported as integrated values (area x density of band) and expressed as a percentage compared with the mean value in controls (100%) for each blot and are representative of all samples from both GD and control rats.

Renal Cortex
In response to fluid deprivation, there were no significant changes by immunoblotting in the renal cortex protein abundance of NHE3, the Na-K-2Cl co-transporter, Na-K-ATPase {alpha}1 and {beta}1 subunit and Gs{alpha} subunit, or AQP1 or 2 in the GD as compared with the control rats (Table 3).


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  		Table 3. Summary of densitometry of immunoblots from study groups with nonsignificant differences in response to 36-h period of water deprivationa

 
Outer Medulla
In the more hypoxic outer medullary region of the kidney, there were several significant differences between the GD and control rats with fluid deprivation. Protein abundance of the Na-K-2Cl co-transporter, Na-K-ATPase {alpha}1 and {beta}1 subunits, and NHE3 was significantly decreased in the GD as compared with control rats (Figure 2). The Gs{alpha} protein expression was also significantly decreased in the outer medulla in the GD as compared with control rats (88 ± 3 versus 100 ± 1% control mean; P < 0.01) as was the AQP2 water channel (95 ± 1 versus 100 ± 1; P < 0.01). There were no differences in AQP1 and 3 or UT-A2.



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  		Figure 2. GD rats demonstrated significant decreases in the outer medulla protein abundance of the Na-K-2Cl co-transporter (A), Na-K-ATPase {alpha}1 subunit (B), Na-K-ATPase {beta}1 subunit (C), and NHE3 (D) as compared with CTL rats.

 
Inner Medulla
In the inner medulla, there were also significant differences in expression of several proteins between GD and control rats. AQP1 and 2 both were significantly decreased in the GD as compared with the control rats (Figure 3, A and B). There were, however, no differences in AQP3 and 4 (Table 3).



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  		Figure 3. GD rats demonstrated significant decreases in the renal inner medulla protein abundance of aquaporin 1 (AQP1; A), AQP2 (B), and urea transporter A1 (UT-A1; C).

 
UT-A2 protein expression was no different between the outer and inner medulla (Table 3), but UT-A1 was significantly decreased in the inner medulla of the GD as compared with the control rats (Figure 3C). In Figure 4 is shown the diminished UT-A by immunohistochemistry in the base of the inner medulla in the GD rats. This difference was not evident in the tip of the inner medulla. In Figure 5 is demonstrated the diminished immunohistochemistry staining for AQP2 in the inner medulla in GD rats. In fact, this decrease in AQP2 seems somewhat greater than that observed by Western analysis. We do not have an explanation for the modest quantitative difference.



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  		Figure 4. Glucocorticoid deficiency was associated with diminished UT-A as assessed by immunohistochemistry in inner medulla base (IM base; B versus D) but not in terminal inner medulla (IM tip; A versus C) as compared with controls (CTL). Magnification, x400.

 


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  		Figure 5. GD rats (B) demonstrated diminished staining for AQP2 in inner medulla on immunohistochemical studies as compared with CTL (A). Negative control (NC; C) stained with secondary antibody only. Magnification, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our study, GD rats were demonstrated to have a significant diminution in maximal urinary osmolality during 36 h of fluid deprivation as compared with the glucocorticoid-replete animals. The systemic hemodynamic alterations that accompanied this defect in maximal urine concentration included a decrease in cardiac output and MAP. A decrease in myocardial contractility has been reported in the presence of glucocorticoid deficiency (21–23). The response of the peripheral vasculature to catecholamines has also been demonstrated to be dependent on glucocorticoid hormones (24). In that regard, the GD rats were shown in this study not to demonstrate the increase in peripheral vascular resistance that normally occurs with a decrease in cardiac output and a fall in MAP. Thus, the GD state was shown to exhibit an effect at both the cardiac and the peripheral vascular levels. Despite the lower MAP in the GD rats, the creatinine clearance, an assessment of GFR, was no different from that observed in the glucocorticoid-replete animals. This observation was no doubt due to the preservation of autoregulation of GFR at the MAP of 73 mmHg observed in the GD rats.

Although a decline in GFR was not involved in the impairment of renal water conservation, there was considerable evidence that several nephron factors that are important to normal urinary concentration were perturbed in the GD state. The primary component of the countercurrent concentrating mechanism is the active reabsorption of sodium chloride in the water-impermeable portion of the medullary thick ascending limb by the Na-K-2Cl co-transporter. In the GD animals, there was a significant downregulation of the protein expression of the Na-K-2Cl co-transporter in the outer medulla. The outer medulla is a relatively hypoxic area of the nephron as compared with the renal cortex, and thus the observed decrease in renal perfusion pressure in the GD animals could have contributed to the diminished expression of the medullary Na-K-2Cl co-transporter in addition to any direct nephron effect of the absence of glucocorticoid hormone. Support for this possibility is that the protein expression of several transporters, including the Na-K-2Cl co-transporter, Na-K-ATPase {alpha}1 and {beta}1 subunits, and NHE3, were not altered during glucocorticoid deficiency in the oxygen-rich renal cortex.

The observed diminution in protein expression of the Na-K-ATPase {alpha}1 and {beta}1 subunits in the outer medulla would be expected to decrease vectorial sodium transport across the basolateral membrane of the water-impermeable segment of the thick ascending limb and thus further impair the countercurrent concentrating mechanism. Some of the diminished NHE3 in the outer medulla with glucocorticoid deficiency no doubt occurred in the proximal tubule, but there is also histochemical evidence for expression of NHE3 in medullary thick ascending limb of the rat (25). A decrease in sodium-hydrogen exchange in the thick ascending limb also could contribute to impairment of the countercurrent concentrating mechanism.

There are additional factors involved in maximal urinary concentration that were examined in our study. AQP1 knockout mice (26) and humans without AQP1 (27) are known to exhibit defects in urinary concentration. AQP1 water channels in the thin descending limb of Henle play an important role in water abstraction, and the resulting increase in luminal sodium chloride concentration toward the tip of the inner medulla therefore is critical to the intact countercurrent concentrating mechanism. In our study, the expression of AQP1 water channels was diminished in the inner medulla of the GD rats.

The interstitial osmotic gradient that is created in the medulla by the countercurrent concentrating mechanism involves not only sodium chloride but also accumulation of urea. Stress doses of exogenous glucocorticoid hormone (100 µg/kg per d of dexamethasone), which exceed the physiologic doses (12 µg/kg per d) used in this study, have been shown to downregulate the UT-A1 protein abundance in the inner medulla (28). High endogenous levels of glucocorticoid hormone in the untreated diabetic rat have also been associated with a similar UT-A1 protein repression (29). In this study, adrenalectomized rats that received mineralocorticoid but not glucocorticoid replacement demonstrated a decrease in UT-A1 protein expression in the inner medulla. UT-A2 expression was not altered in either the outer or the inner medulla. The glucocorticoid deficiency was also associated with an increase in the fractional excretion of urea, a finding consonant with the decrease in UT-A1 protein expression. Immunohistochemistry studies demonstrated that the decrease in the UT-A1 protein was primarily in the basal portion of the inner medulla of the glucocorticoid rat. This is particularly important because replacement of dexamethasone (100 µg/kg per d) has been shown to decrease the vasopressin-responsive urea transporter only in the terminal portion of the inner medulla of adrenalectomized rats that did not receive replacement of mineralocorticoid (28). In our study, the decrease in UT-A1 in the GD rats would be expected to diminish urea transport and recycling between vasa recta and the thin descending limb of Henle’s loop, an important factor in the maximal generation of the medullary osmotic gradient. The decrease in inner medullary protein abundance for UT-A1 was associated with diminished UTA by immunohistochemistry.

Maximal urine concentration also involves the integrity of membrane water channels. In addition to AQP1, the AQP2, 3, and 4 water channels in the mammalian collecting duct are important in the concentration of urine. In our study, the observed decrease in maximal urinary osmolality and increase in urine flow in the GD rats during fluid deprivation are compatible with a downregulation or decreased trafficking of the AQP2 water channels to the apical surface of the collecting duct, as has been shown in several models of acquired nephrogenic diabetes insipidus (e.g., hypokalemia, hypercalcemia, lithium administration) (30–32). Although there were no differences in AQP3 and 4 between GD and control rats, there was a decrease in AQP2 expression in both the outer and the inner medulla in the absence of glucocorticoid hormone. This alteration in AQP2 between the GD and glucocorticoid-replete states during fluid deprivation occurred in the absence of differences in plasma vasopressin. The action of vasopressin with glucocorticoid deficiency, however, could have been blunted as a result of the observed decrease in the outer medulla of Gs{alpha}, an important factor in vasopressin signaling.

The medullary osmolality was diminished in the GD state, a finding supportive of an impairment in the countercurrent concentrating mechanism. Although glucocorticoid deficiency was associated with a decrease in AQP2, there seemed to be osmotic water equilibration between the terminal medullary collecting duct fluid and the medullary interstitium, because no significant difference was observed between the medullary and urine osmolality.

In summary, an impairment in maximal urinary concentration was demonstrated in adrenalectomized rats that received replacement of aldosterone but not glucocorticoid. This defect in urinary concentration in the GD state was associated with diminished protein expression of critical factors in the countercurrent concentrating mechanism, including the Na-K-2Cl co-transporter, Na-K-ATPase {alpha}1 and {beta}1, AQP1, and UT-A1. These defects associated with glucocorticoid deficiency occurred in the relatively hypoxic medulla in association with a decrease in cardiac output and renal perfusion pressure. There was also a significant decrease in AQP2 but not AQP3 or 4 water channels during glucocorticoid deficiency. However, because equilibration of fluid between medullary interstitium and urinary osmolality was observed, a multifactorial impairment in the countercurrent concentrating mechanism seems to be the predominant defect in renal water conservation during glucocorticoid deficiency.


   Acknowledgments
 
This work was supported by the National Institutes of Health (DK19928). Y.-C.C. was supported by a grant from Chang Gung Memorial Hospital (Taipei, Taiwan).


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 16, 2004. Accepted for publication July 13, 2005.


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