Glucocorticoids mediate a decrease in AVP-regulated urea transporter in diabetic rat inner medulla

Janet D. Klein, S. Russ Price, James L. Bailey, Joely D. Jacobs, and Jeff M. Sands

Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Providing glucocorticoids to adrenalectomized (Adx) rats results in downregulation of the vasopressin (AVP)-regulated urea transporter (VRUT) in the renal inner medullary (IM) tip. To examine the physiological relevance of this response, we studied rats with uncontrolled diabetes mellitus induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We measured VRUT protein in extracts from the IM tip or base of pair-fed control and diabetic rats by Western analysis using an antibody to rat VRUT. In the IM tip, VRUT was significantly reduced by 39% in diabetic compared with control rats. In the IM base, there was no significant difference between diabetic and control rats. To determine whether the decrease in VRUT in the IM tip was mediated by glucocorticoids, the experiment was repeated using the following three groups of rats: 1) Adx alone, 2) Adx + STZ, and 3) Adx + STZ + replacement with a physiological dose of glucocorticoid. There was no significant difference in VRUT between Adx and Adx + STZ rats. However, VRUT was significantly reduced by 32% in the IM tip of glucocorticoid-treated Adx + STZ rats compared with control Adx + STZ rats. We conclude that glucocorticoids regulate the abundance of VRUT protein independently of insulin in diabetic rats.

inner medullary collecting duct; urine concentration; diabetes mellitus; vasopressin

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

A VASOPRESSIN-REGULATED urea transporter (VRUT) is normally expressed in the rat terminal inner medullary collecting duct (IMCD) (16). Knepper and colleagues (9) showed that administering glucocorticoids to adrenalectomized (Adx) rats increased their fractional excretion of urea, and they concluded that the mechanism involved a decrease in tubular reabsorption of urea. We prepared a VRUT antibody and used it to show that administering glucocorticoids to an Adx rat downregulates urea transport in the terminal IMCD and the expression of the VRUT protein in the inner medullary tip (11).

Diabetes mellitus is a common clinical disorder characterized by both increased glucocorticoid production and increased urea excretion (1, 2, 4, 8). Given our previous results (11), it is possible that the increase in urea excretion in diabetes mellitus is due to glucocorticoid-mediated downregulation of VRUT expression in the inner medulla. Accordingly, the present studies examined the effects of streptozotocin (STZ)-induced diabetes mellitus on the expression of VRUT protein and the role of glucocorticoids in the regulation of its expression in this setting.

    METHODS
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Introduction
Methods
Results
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References

Animal preparation. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 175-200 g received free access to 23% protein rat diet and water for at least 3 days after delivery. Rats were given an injection of STZ (125 mg/kg body wt prepared fresh in 0.1 M citrate buffer, pH 4.0; Pfanstiehl Laboratories, Waukegan, IL) into a tail vein under ether anesthesia at 7 AM. Subsequently, the rats were fed 23% protein chow ad libitum, and the quantity of food eaten was measured. Vehicle-injected control rats were pair-fed the same amount of food. At 6 PM on the night before an experiment, food was removed from both groups of rats to eliminate the variability in responses related to differences in food absorption. After anesthesia with ketamine (Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (Miles Agricultural Division, Shawnee Mission, KS), arterial blood was collected to measure blood glucose (One Touch Profile Diabetes Tracking Kit; Lifescan, Milpitas, CA), bladder urine was collected to measure urine osmolality (model 5500 vapor-pressure osmometer; Wescor, Logan, UT) and glucose and ketones (Ames N-Multistix SG; Miles, Elkhart, IN), and the kidneys were removed (14).

In a separate protocol, rats underwent Adx and were allowed to recover for 7 days (11). Some Adx rats were injected with STZ as described above. A third group of Adx rats was injected with STZ and was injected with dexamethasone (Elkins-Sinn, Cherry Hill, NJ) subcutaneously twice a day at a dose (1 µg/100 g body wt) designed to approximate physiological glucocorticoid levels (7). The quantity of 23% protein chow eaten by the Adx, STZ-treated rats was measured, and control vehicle-injected Adx rats and the Adx, STZ-treated, dexamethasone-treated rats were pair-fed the same amount of food. At 6 PM the night before an experiment, food was removed from all three groups of rats, and the kidneys were harvested the next morning as described above.

Western blot analysis. Rat kidney inner medullas were dissected into two regions: base and tip, corresponding to the location of the initial and terminal IMCD (5, 6), respectively, as previously described (3, 11, 15). Tissue from both kidneys of a single rat was pooled and placed into an ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, 1 mg/ml leupeptin, and 2 mg/ml phenylmethylsulfonyl fluoride), homogenized, and diluted 1:1 with 1% sodium dodecyl sulfate (SDS) for Western analysis of total cell lysate (11, 13, 15). Total protein in each sample was measured by the Bradford method (Bio-Rad, Richmond, CA). Proteins (15 µg/lane) were size-separated by SDS-polyacrylamide gel electrophoresis on 10% Laemmli gels, electroblotted to polyvinylidene difluoride membranes (PVDF; Gelman Scientific, Ann Arbor, MI), and incubated for 30 min at room temperature with blocking buffer, which was 5% nonfat dry milk suspended in tris(hydroxymethyl)aminomethane (Tris)-buffered saline (TBS; 20 mM Tris · HCl, 0.5 M NaCl, pH 7.5) (11).

The Western blots were probed with the following primary polyclonal antibodies diluted 1:1,000 to 1:2,000 in TBS with 0.5% Tween 20: 1) our affinity-purified antibody "C" to the rat VRUT (11); or 2) an antibody to rat aquaporin-2 (AQP2), the rat vasopressin-regulated water channel (generous gift from Dr. H. William Harris, Harvard University) (12). Blots were incubated with primary antibody overnight at 4°C, then washed three times in TBS-Tween. Blots were then incubated with horseradish peroxidase-linked goat anti-rabbit immunoglobulin G at a dilution of 1:4,000 to 1:5,000 (Amersham, Arlington Heights, IL) for 2 h at room temperature and washed twice with TBS-Tween, and the bound secondary antibody visualized using enhanced chemiluminescence (ECL kit, Amersham). Laser densitometry was used to quantitate the ECL signal. Results are expressed as units per milligram protein loaded. In all cases, parallel gels were stained with Coomassie blue and showed uniformity of loading (data not shown).

Statistics. Data are presented as means ± SE, where n indicates the number of rats studied. To test for statistically significant differences between two groups, a paired Student's t-test was used. To test for statistically significant differences among three groups, an analysis of variance was used followed by a multiple comparison, protected t-test (17).

    RESULTS
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Methods
Results
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References

Diabetic rats. The rats injected with STZ had significantly higher values of blood glucose (Table 1A) and urine osmolality (control, 212 ± 35; STZ treated, 598 ± 73 mosmol/kgH2O; P < 0.01). All diabetic rats had >= 2 g/dl glucosuria, whereas none of the control rats had glucosuria. As reported, the diabetic rats lost weight during the 3 days following injection with STZ, and 42% had ketones in their urine (14). The amount of corticosterone measured in a 24-h collection was greater than sixfold higher in diabetic rats than in control rats (S. R. Price and W. E. Mitch, unpublished observations).

                              
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Table 1.   In vivo parameters

After 3 days of diabetes, the 97-kDa VRUT protein (13) was 39% lower in the inner medullary tip of the kidneys of diabetic rats compared with control rats (n = 6, P < 0.05; Fig. 1). To determine whether this effect was due to a generalized depression of transporter proteins, we measured AQP2 protein. In contrast to VRUT, there was no significant difference in AQP2 protein in the kidneys of control and diabetic rats (n = 5; Fig. 2).


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Fig. 1.   A: representative immunoblot (Western) of protein from inner medullary tip of control and from streptozotocin (STZ)-treated diabetic (DM) rats after probing with our vasopressin-regulated urea transporter (VRUT) antibody (11). Each lane shows protein from an individual rat. Band at 97 kDa is the VRUT protein (13). It is significantly decreased in diabetic rats compared with control rats. The 2 lower molecular mass bands are specifically recognized by the antibody, although the identity of the proteins that correspond to these 2 smaller bands is unknown. B: summary of laser densitometric analysis. The 97-kDa VRUT protein in the inner medullary tip from diabetic rats is significantly decreased compared with control rats. * P < 0.05; n = 6.


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Fig. 2.   A: representative immunoblot (Western) of protein from inner medullary tip from control (C) and STZ-treated diabetic (DM) rats, probed with an antibody to AQP2 (12). There is a band at 29 kDa and a broad band at 35-45 kDa. Each lane shows protein from an individual rat. B: summary of laser densitometric analysis. There is no significant difference in the level of AQP2 protein in proteins isolated from the inner medullary tip of control and diabetic rats; n = 5.

Next, we measured VRUT protein in the inner medullary base of the kidney. There was no significant difference in VRUT protein between control and diabetic rats (n = 6; Fig. 3).


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Fig. 3.   A: representative immunoblot (Western) of protein from inner medullary base of control (C) and from STZ-treated diabetic (DM) rats, probed with our VRUT antibody (11). Each lane shows protein from an individual rat. There is no significant difference in the band at 97 kDa between control and diabetic rats. B: summary of laser densitometric analysis. There is no significant difference in VRUT protein in the inner medullary base between control and diabetic rats; n = 6.

Adx rats. If the decrease in VRUT protein in the renal inner medullary tip of diabetic rats requires glucocorticoids, then Adx should block the decrease in VRUT protein, and replacing glucocorticoids would restore it. We performed this experiment using three groups of Adx rats. The Adx rats injected with STZ had significantly higher values of blood glucose and weight loss than control Adx rats (Table 1B).

There was no significant difference in VRUT protein in the inner medullary tip of the kidney in a control group of Adx rats and diabetic-Adx rats (n = 6; Fig. 4). However, VRUT protein was 32% lower in the inner medullary tip of the kidneys of diabetic-Adx rats treated with dexamethasone compared with the findings in the control Adx rats or diabetic-Adx rats (n = 6, P < 0.05; Fig. 4).


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Fig. 4.   A: representative immunoblot of proteins isolated from inner medullary tips of adrenalectomized rats (Adx); adrenalectomized, STZ-treated rats (Adx + DM); and adrenalectomized, STZ-treated, dexamethasone-replaced rats (Adx + DM + Dex) after probing with our VRUT antibody (11). Each lane shows protein from an individual rat. There is no significant difference in the 97-kDa band between Adx and Adx + DM rats. However, the band at 97 kDa is significantly decreased in Adx + DM + Dex rats compared with Adx or Adx + DM rats. B: summary of laser densitometric analysis. There is no significant difference in VRUT protein in the inner medullary tip between Adx and Adx + DM rats. However, VRUT protein is significantly reduced in Adx + DM + Dex rats compared with Adx or Adx + DM rats. * P < 0.05; n = 6.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In rats injected with STZ, there is a decrease in the level of VRUT protein in the renal inner medullary tip. This response is mediated by glucocorticoids rather than other complications of diabetes (e.g., glucosuria, hyperglycemia, insulinopenia), since Adx prevents the decrease in VRUT protein but glucocorticoid replacement (7) restores the response. This finding is consistent with our previous study, which showed that providing glucocorticoids to Adx rats reduces the level of VRUT protein in the inner medullary tip of the kidney (11). These results extend our previous findings (11). In those studies, providing glucocorticoids to Adx rats decreased urea transport in terminal IMCDs and downregulated the abundance of VRUT protein, and since there was no change in mRNA abundance, we concluded that a translational or posttranslational mechanism accounted for the response (11). The present studies show that the level of VRUT protein is decreased in the inner medullary tip of rats with uncontrolled diabetes mellitus, a clinically relevant condition associated with increased glucocorticoid production and increased urea excretion (1, 2, 4, 8). Furthermore, this decrease in VRUT protein is dependent upon glucocorticoids rather than insulin, since Adx prevents this response in diabetic rats. Neither this nor our previous study (11) determines whether the decrease in VRUT protein occurs by a direct effect of glucocorticoids or as a secondary response to glucocorticoid-induced physiological or pathophysiological changes.

The Adx and diabetic-Adx rats had lower values for blood glucose than the adrenal intact control and diabetic rats, respectively, raising the possibility that VRUT protein expression is affected by the level of glycemia. However, the reduction in VRUT protein appears to be independent of the level of glycemia, since 1) the diabetic-Adx rats were hyperglycemic compared with control Adx rats, yet there was no significant difference in the level of VRUT protein expression between the two groups of rats; and 2) our previous study showed no difference in blood glucose between sham-operated control rats, Adx rats, and dexamethasone-treated Adx rats (11). Thus our data suggest that the reduction in VRUT protein in diabetic rats is not due to hyperglycemia.

Although STZ can be nephrotoxic, several findings indicate the reduction in VRUT protein is a specific consequence of diabetes rather than a nonspecific response to STZ. First, there was no difference in AQP2 protein between control and diabetic rats in the tip of the inner medulla. Second, there was no difference in VRUT protein between control Adx and diabetic-Adx rats in the tip of the inner medulla. Third, there was no difference in VRUT protein between control and diabetic rats in the base of the inner medulla.

The rats in this study lost 20% of their body weight in 3 days (14). This weight loss is consistent with the highly catabolic condition of rats or humans with poorly controlled diabetes mellitus. We recently showed that STZ-treated rats have an increase in muscle protein wasting due to activation of the ubiquitin-proteosome pathway mediated by an increase in transcription of the ubiquitin gene (14). The increase in muscle proteolysis is likely to increase urea production and the need to increase urea excretion. Indeed, several studies have shown a marked increase in urea excretion in both humans and rats with uncontrolled diabetes mellitus (1, 2, 4, 8). The collecting duct must balance the need to reabsorb urea to maintain the inner medullary urine concentrating ability with the need to excrete urea and remove nitrogen from the body (10). We speculate that glucocorticoids, mediators of catabolism, act to decrease VRUT protein to reduce urea reabsorption across the terminal IMCD and increase urea excretion.

    ACKNOWLEDGEMENTS

We thank Dr. William E. Mitch (Emory University) for helpful discussions throughout the course of these experiments and critical reading of this manuscript and Dr. H. William Harris (Harvard University) for generously providing the antibody to rat AQP2.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-41707 and P01-DK-50268 (to J. M. Sands) and R01-DK-50740 (to S. R. Price).

Portions of this work have been published in abstract form (J. Am. Soc. Nephrol. 7: 1268, 1996; and FASEB J. 11: 24, 1997) and have been presented at the 29th Annual Meeting of the American Society of Nephrology, November 3-6, 1996, New Orleans, LA, and at Experimental Biology '97, April 6-9, 1997, New Orleans, LA.

Address for reprint requests: J. M. Sands, Emory Univ. School of Medicine, Renal Division, WMRB Rm. 338, 1639 Pierce Drive NE, Atlanta, GA 30322.

Received 13 May 1997; accepted in final form 28 August 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Renal Physiol 273(6):F949-F953
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