Molecular Mechanisms of Impaired Urinary Concentrating Ability in Glucocorticoid-Deficient Rats Yung-Chang Chen,*† Melissa A. Cadnapaphornchai,*‡ Sandra N. Summer,* Sandor Falk,* Chunling Li,* Weidong Wang,* and Robert W. Schrier* Departments of *Medicine and ‡Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado; and †Division of Critical Care Nephrology, Chang Gung Memorial Hospital, Taipei, Taiwan 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␣ 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. J Am Soc Nephrol 16: 2864–2871, 2005. doi: 10.1681/ASN.2004110944 he ability to conserve water during periods of fluid limb, creates the osmotic driving force for passive water reab- deprivation is an important function of the kidney. In sorption across the collecting duct. There are also roles for other T both humans and experimental animals, adrenal insuf- water channels, including aquaporins 1, 3, and 4, and for urea ficiency has been associated with several alterations in renal transporters in urinary concentration. This study was under- function, including impairment of urinary diluting and concen- taken to define the effect of glucocorticoid deficiency on these trating capacity (1–7). Isolated glucocorticoid deficiency has various molecular events during fluid deprivation in the rat. also been associated with impaired urinary concentration (8,9). However, the mechanisms of this defect at the cellular and Materials and Methods molecular levels have not been defined. Animal Model In this study, glucocorticoid-deficient (GD) rats were com- The study protocol was approved by the University of Colorado pared with glucocorticoid-replete rats with respect to their Institutional Animal Care and Use Committee. Male Sprague-Dawley capacities to concentrate the urine. Critical components of uri- rats that weighed 175 to 200 g were allowed to acclimate to Denver’s nary concentration in response to fluid deprivation include altitude (1500 m) for 1 wk before any experimental protocols. All release of the antidiuretic hormone arginine vasopressin (AVP) animals underwent acclimation to metabolic cages for a continuous 5-d and upregulation of the abundance of aquaporin-2 (AQP2) period before initiation of study. The animals were housed individually water channels in the principal cells of the collecting duct. in metabolic cages and exposed to a 12-h light-dark cycle and constant Moreover, activation of the countercurrent concentrating mech- ambient temperature. Eighteen rats were divided equally into each of anism, which is initiated by the sodium-potassium-2 chloride two study groups: GD and control. Under anesthesia with ketamine (40 (Na-K-2Cl) co-transporter in the water-impermeable ascending 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 Received November 16, 2004. Accepted July 13, 2005. (Research Plus, Bayonne, NJ) at a dose calculated to deliver 17 ␮g/kg Published online ahead of print. Publication date available at www.jasn.org. per 24 h into the peritoneal cavity were implanted into GD rats (10). Control rats received combined treatment with osmotic minipumps Address correspondence to: Dr. Robert W. Schrier, Division of Renal Diseases that contained aldosterone plus subcutaneous injections of dexameth- 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; asone (Research Plus) dissolved in peanut oil at a dose of 12 g/kg per E-mail: [email protected] d starting immediately after adrenalectomy. This dose of dexametha- Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1610-2864 J Am Soc Nephrol 16: 2864–2871, 2005 Impaired Urinary Concentrating Ability in Glucocorticoid-Deficient Rats 2865 sone has been reported to maintain normal weight gain, GFR, and medulla; and UT-A1 and AQP4 in the inner medulla. SDS-PAGE was fasting plasma glucose and insulin levels in adrenalectomized rats (11). performed on 8% acrylamide gels for the Na-K-2Cl co-transporter, For this study, we elected to use hormone-replaced adrenalectomized Na-K-ATPase ␣1 subunit, and UTA and on 12% acrylamide gels for rats as controls, rather than intact (unaltered) animals. Our studies AQP, NHE3, Na-K-ATPase ␤1 subunit, and Gs␣ subunit proteins. After (data not shown) demonstrated that the protein abundance of inner transfer by electroelution to polyvinylidene difluoride membrane (Mil- medulla urea transporter A1 (UT-A1) was similarly diminished in the lipore, Bedford, MA), blots were blocked overnight with 5% nonfat intact rats and in our controls. dried milk in PBS(Ϫ) and then probed with the respective antibodies After adrenalectomies, all rats were pair-fed with plain powdered rat for 24 h at 4°C. After washing with buffer that containing PBS(Ϫ) with chow (Harlan Teklad Bioproducts, Indianapolis, IN) 15 g/d. Drinking 0.1% Tween 20 (J.T. Baker, Phillipsburg, NJ), the membranes were water was provided ad libitum. All animals were maintained in meta- exposed to secondary antibody for1hatroom temperature. Subse- bolic cages for the duration of the study to assess accurately daily food quent detection of the specific proteins was carried out by enhanced intake, water intake, and urine output. chemiluminescence (Amersham, Arlington Heights, IL) according to On day 7 after adrenalectomies, echocardiography was performed the manufacturer’s instructions. Prestained protein markers were used using a GE Vingmed System 5 imaging tool (GE, Horten, Norway) for for molecular mass determinations. Densitometric results were re- small rodents with a 10-MHz probe. The animals were anesthetized for ported as integrated values (area ϫ density of band) and expressed as echocardiography with ketamine and xylazine in doses as described a percentage compared with the mean value in controls (100%). Mem- above. Cardiac output was calculated via measurement of the diameter branes were stained with Coomassie blue to ensure equal loading. For of the left ventricular outflow tract (LVOT), the flow through the each gel, an identical gel was run in parallel and subjected to Coomas- outflow tract (VTI), and the heart rate (HR) by the formula, 0.785 ϫ sie staining to verify identical protein loading. Blots shown in the LVOT2 ϫ VTI ϫ HR. The right femoral artery then was catheterized Results section are representative of the results obtained from all sam- with a polyethylene tube (PE-50; Intramedic, Clays Adams, Parsippany, ples. Densitometry as shown in the Results section reflects means Ϯ NJ), and BP was measured using a Transpac disposable transducer SEM densitometry of all 18 samples. (Abbott Critical Care Systems, Salt Lake City, UT) connected to a Transonic Systems T106 BP monitor (Ithaca, NY). BP was analyzed Antibodies using WinDaq software (Dataq Instruments, Akron, OH). Cardiac out- Antibodies to AQP2, AQP3, AQP4, NHE3, UT-A1, and UT-A2 have put was factored by body weight and expressed as cardiac index (CI; been characterized previously (15–19). Anti–Na-K-ATPase ␣1 and ␤1 ml/min per 100 g). Total peripheral resistance (mmHg/min per ml/100 antibodies were obtained from Upstate Biotechnology (Lake Placid, g) was calculated by dividing mean arterial pressure (MAP) by CI. NY). Antibodies to AQP1 and the Na-K-2Cl co-transporter were ob- Stroke volume (ml/beat per 100 g) was obtained by dividing CI by HR tained from Chemicon International, Inc. (Temecula, CA). Anti-Gs␣ (12,13). The catheter was removed. Animals were allowed to recover antibody was obtained from Calbiochem-Novabiochem (San Diego, and then were returned to metabolic cages. CA). Two days after echocardiography, all animals