Urea may regulate urea transporter protein abundance during osmotic diuresis
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《美国生理学杂志》
Renal Division, Department of Medicine, and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
Department of Pediatrics, The Catholic University of Korea, Seoul, Republic of Korea
ABSTRACT
Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.
sodium-potassium-2 chloride cotransporter; diabetes mellitus
THE RENAL MEDULLA IS THE LOCATION in which water excretion is controlled through the production of concentrated or dilute urine. Several solute transport proteins play a major role in the urinary concentrating mechanism, including urea transporters and the Na+-K+-2Cl– cotransporter (NKCC2/BSC1). Among the urea transporters, UT-A1 is expressed in the inner medullary collecting duct and is important for vasopressin-regulated urea reabsorption (reviewed in Ref. 21). UT-A2 and UT-B are expressed in the thin descending limb and descending vasa recta, respectively, and are important for intrarenal urea recycling (reviewed in Ref. 21). NKCC2/BSC1 is expressed in the thick ascending limb of the loop of Henle and is responsible for the active reabsorption of NaCl that drives the single effect to concentrate urine (reviewed in Ref. 22).
Several studies show that UT-A1, as well as NKCC2/BSC1 and aquaporin-2 (AQP2), protein abundances increase after 5 days of uncontrolled diabetes mellitus due to streptozotocin (2, 10, 11, 19, 27). We showed that UT-A1 protein abundance does not change in streptozotocin-treated Brattleboro rats, indicating that vasopressin is necessary for the increase in UT-A1 protein abundance because Brattleboro rats lack vasopressin (11). When we administered vasopressin to Brattleboro rats and then induced diabetes mellitus, UT-A1 abundance increased. Interestingly, UT-A1 abundance was increased more in vasopressin-infused diabetic Brattleboro rats than in Brattleboro rats receiving vasopressin alone. These findings imply that vasopressin is necessary but cannot be the signal to increase UT-A1 abundance because the two groups of Brattleboro rats received the same amount of vasopressin (11).
Urea is the major urinary solute in mammals, comprising 40–50% of total urinary solute in rats on a normal diet. Rats with streptozotocin-induced diabetes mellitus do have an increase in absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased (to 20%) due to the marked increase in glucose excretion (10). The ongoing osmotic diuresis due to nonreabsorbable glucose causes water to be retained in the tubule lumen, which dilutes the urine urea concentration. Several urea-specific signaling pathways have been identified in cultured mIMCD3 cells and the renal medulla (reviewed in Refs. 3 and 28), suggesting the possibility that changes in the percentage or concentration of urea in the urine, tubular fluid, or medullary interstitium could be a factor that regulates UT-A1 protein abundance. Consistent with this possibility, urea has been shown to inhibit Na+-K+-2Cl– cotransport in medullary thick ascending limb cells (9). In the present study, we tested the hypothesis that an increase in any urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus-induced glucose diuresis, whereas an increase in total urinary solute due to urea itself would not result in an increase in UT-A1. If this were true, then it would suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability. To test our hypothesis, we measured the protein abundance of UT-A1 during several models of osmotic diuresis in which the percentage of urea in total urinary solute was varied. We also measured the abundances of UT-A2, UT-B, and NKCC2/BSC1 to evaluate the specificity of any changes in UT-A1.
METHODS
Animal preparation. All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 125–200 g, received free access to a standard powdered rat chow (Testdiet 5001, Purina) containing 23% protein and 1.05% NaCl (standard diet). All rats received free access to water throughout the study. Osmotic diuresis due to glucose, NaCl, and/or urea was induced as follows.
To induce glucose diuresis, rats were made diabetic by injecting streptozotocin (60 mg/kg body wt prepared fresh in 0.1 M citrate buffer, pH 4.0, Sigma, St. Louis, MO) into a tail vein at 7 AM (10, 11, 13). Diabetes mellitus was confirmed by measuring a spot urine glucose at 24 and 48 h after streptozotocin injection (Ames N-Multistix SG, Miles, Elkhart, IN). Diabetic rats were fed the standard diet throughout the study. An additional group of rats was made diabetic, and 10% urea was added to the standard powdered diet to keep the percentage of urea in total urinary solute at the control level.
To induce NaCl diuresis, rats were fed a diet containing a total of 4 or 2.5% NaCl by adding 3 or 1.5% NaCl, respectively, to the standard powdered diet (which contains 1.05% NaCl). An additional group of rats was fed 4% NaCl and 3% urea to keep the percentage of urea in total urinary solute at the control level. NaCl was chosen as the osmolyte in this study because introducing mannitol or similar osmolytes that are not absorbed by the gastrointestinal tract would result in osmotic diarrhea rather than osmotic diuresis. In addition, it is difficult to deliver sufficient amounts of these sugars parenterally to approximate the glucose levels that are produced in diabetes mellitus, whereas NaCl can readily be delivered and appropriate levels achieved.
To induce urea diuresis, rats were fed the standard diet to which 5 or 20% urea was added. Each osmotic diuresis group (n = 6 rats) was compared with its own control group (n = 4–6 rats), which was studied in parallel. Because our previous studies showed that UT-A1 protein abundance increases consistently from 10–20 days after streptozotocin-induced diabetes mellitus (10, 11), osmotic diuresis was induced for 15–20 days in all protocols, except that 4% NaCl diuresis was induced for both 5 and 15 days to distinguish whether changes seen at 15 days were temporally distinct as they are in diabetes mellitus. Two days before death, rats were put into metabolic cages and a 24-h urine collection was obtained to measure urine volume, osmolality (model 5500 vapor pressure osmometer, Wescor, Logan, UT), and urea concentration (Infinity BUN reagent, Sigma). After 15–20 days of osmotic diuresis, rats were killed and trunk blood was collected to measure urea nitrogen (BUN) and osmolality. Blood and urine glucose concentrations were measured only in the streptozotocin-treated rats (One Touch Profile Diabetes Tracking Kit, Lifescan, Milpitas, CA). The absence of glucosuria in the other groups was confirmed using urine dip-sticks. Osmolar clearance (Cosm) and solute free water reabsorption (SFWR) were calculated as follows: Cosm = Uosm/Posm x Uvol; SFWR = Cosm – Uvol, where Uosm is urine osmolality, Posm is plasma osmolality, and Uvol is urine volume.
Tissue urea concentration. Kidneys were removed, and the medulla was dissected into outer medulla, inner medullary base, and inner medullary tip as previously described (10, 11). These tissue pieces from one kidney per rat were weighed and processed to determine interstitial urea concentration essentially as described by Schmidt-Nielsen and colleagues (23). Briefly, tissue (10 mg/piece) was weighed wet, dried over dessicant overnight, reweighed, and then urea was extracted from the dried tissue into an aqueous solution that was assayed for urea nitrogen (ThermoTrace, Melbourne, Australia). The remainder of the extract was dried under vacuum, rehydrated in a small volume, and the osmolality was determined. The contralateral kidney was processed for Western blot analysis.
Western blot analysis. Kidney tissue was placed into ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, 1 μg/ml leupeptin, and 2 mg/ml PMSF), homogenized; then, SDS was added to a final concentration of 1% for Western blot analysis of the total cell lysate (10, 11). Total protein in each sample was measured by a modified Lowry method (Bio-Rad DC protein assay reagent, Bio-Rad, Richmond, CA). Proteins (10 μg/lane) were size separated by SDS-PAGE using 7.5, 10, or 15% polyacrylamide gels, blotted to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI), and Western blotting was performed as described previously (10, 11).
Western blots were probed with antibodies (diluted in TBS/Tween) to 1) the UT-A1 and UT-A2 urea transporters (10, 11, 18); 2) the UT-B urea transporter (29); and 3) NKCC2/BSC1 (6, 10–12) and visualized with enhanced chemiluminescence (Amersham, Arlington Heights, IL). Blots were quantified using an imaging densitometer (GS670, Bio-Rad) and Molecular Analyst software (Bio-Rad). In all cases, parallel gels were stained with Coomassie blue to confirm uniformity of loading (data not shown). Results are expressed as arbitrary units per microgram protein.
Statistics. Data are presented as means ± SD (n), where n indicates the number of rats studied. To test for statistically significant differences between two groups, an unpaired Student's t-test was used. To test for statistically significant differences among three or more groups, an analysis of variance was used followed by a multiple-comparison, protected t-test (26).
RESULTS
Physiological parameters. Table 1 shows the urine osmolality, volume, urea, glucose, and other solutes, and plasma urea, glucose, and osmolality. Total urinary solute and urine volume were highest in rats with diabetes mellitus (glucose diuresis) and diabetes mellitus+urea and also increased in the 20% urea diuretic rats (Fig. 1). Urine osmolality was lowest in the two diabetic groups, but also reduced in the 4% NaCl+urea and the 20% urea diuretic rats. Urine urea excretion was highest in diabetes+urea and 20% urea diuretic rats but also increased in diabetic rats.
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Urine urea concentration and the relative amount (percentage) of urea in total urinary solute were decreased in diabetes mellitus and NaCl diuresis and increased in urea diuresis. The percentage of urea in total urinary solute was maintained at the control level in diabetes+urea and in the 4% NaCl+urea diuretic rats, although the urine urea concentration was reduced in these groups compared with control. However, the urine urea concentrations in rats with diabetes+urea and 4% NaCl+urea diuresis were significantly increased compared with rats with diabetes and 4% NaCl diuresis without urea supplementation, respectively.
Effect of diabetes mellitus and NaCl diuresis on UT-A1, UT-A2, UT-B, and NKCC2/BSC1 protein abundances. UT-A1 abundance in both portions of the inner medulla was significantly increased in diabetic rats (255% of control in inner medullary base, 171% of control in inner medullary tip), consistent with our previous results (10, 11), and in rats with 4% NaCl diuresis for 15 days (166% of control in inner medullary base, 162% of control in inner medullary tip) (Fig. 2). It was unchanged in 2.5% NaCl diuretic rats. Because UT-A1 abundance varies with time after streptozotocin injection (10), we tested whether there was a temporal change in UT-A1 after induction of 4% NaCl diuresis. UT-A1 abundance increased at 5 days of 4% NaCl diuresis in the inner medullary base, but not in the inner medullary tip, similar to the pattern of changes in UT-A1 at 5 days of diabetes (10).
In contrast, UT-A2 abundance in the outer medulla was significantly decreased in diabetic rats (66% of control) and in rats with 2.5% NaCl diuresis (72% of control) but was unchanged in 4% NaCl diuresis (Fig. 3) rats. UT-B abundance was unchanged in any portion of the medulla with either diabetes or 4% NaCl diuresis (Fig. 4). NKCC2/BSC1 abundance in the outer medulla (Fig. 5) was significantly increased in both diabetic rats (143% of control) and 4% NaCl diuretic (130% of control) but not in 2.5% NaCl diuretic rats.
Effect of urea feeding on diabetic and 4% NaCl diuretic rats. The data in Figs. 1 and 2 suggest the hypothesis that a decrease in urine urea below a certain level may be a signal that increases UT-A1 abundance in the inner medulla. To test this hypothesis, diabetic and 4% NaCl diuretic rats were fed supplemental urea to restore the percentage of urea in urine solute to control levels (Table 1, Fig. 1). Diabetic rats and 4% NaCl diuretic rats fed urea supplements did not show significant differences in UT-A1 (Fig. 6), UT-A2 (Fig. 7), or UT-B abundances (Fig. 8) compared with control rats, suggesting that restoring the percentage of urea in total urinary solute reverses the diabetes- or 4% NaCl diuresis-induced upregulation of UT-A1 abundance. NKCC2/BSC1 abundance (Fig. 9) was significantly increased in both rats with diabetes+10% urea diuresis (124% of control) and 4% NaCl+3% urea diuresis (121% of control). However, the percentage increases were somewhat smaller than the increases in rats with diabetes or 4% NaCl diuresis alone.
Effect of urea diuresis. UT-A1 abundance was not changed in rats with 5 or 20% urea diuresis in either the inner medullary base or tip (Fig. 6), even though these rats had severe osmotic diuresis. Considering that the urine urea concentration is higher, but urine osmolality is lower, than those of control rats (Table 1, Fig. 1), this suggests that UT-A1 is responding to the urine urea concentration, rather than urine osmolality. In contrast, UT-A2 abundance in the outer medulla was significantly increased with both the 5% urea (163% of control) and 20% urea (155% of control) diuresis (Fig. 7). UT-B abundance in the outer medulla was also significantly increased in both rats with 5% urea (150% of control) and 20% urea (130% of control) diuresis but was unchanged in the inner medullary base (Fig. 8). NKCC2/BSC1 abundance showed a tendency to increase in both rats with 5 and 20% urea diuresis, but the increase was statistically significant only in 20% urea diuretic rats (Fig. 9).
Tissue urea concentration. To determine whether the changes in urine urea reflected tissue urea, we determined tissue urea concentrations in 20% urea diuretic and 4% NaCl diuretic rats. Compared with control rats, tissue urea levels were significantly increased in all medullary regions in the 20% urea diuretic rats (Table 2). There was no difference in the tissue urea content of the 4% NaCl diuretic rats compared with control rats.
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DISCUSSION
UT-A1. The major finding in this study is that UT-A1 protein abundance increases during osmotic diuresis whenever the relative amount (percentage) of urea in total urinary solute decreases. Regardless of whether glucose (diabetes mellitus) or NaCl induced the osmotic diuresis, thereby reducing the percentage of urea in the urine, UT-A1 abundance increased. When osmotic diuresis was induced by urea, the percentage of urea in the urine did not decrease and UT-A1 abundance did not increase.
To further test the role of urine urea, rats undergoing osmotic diuresis due to glucose or NaCl were given urea supplements to prevent the decrease in the percentage of urea in the urine. With the urea-supplemented diets, the percentage of urea in total urinary solute was adjusted so that is was similar to that in control rats. In both of the urea-supplemented groups, UT-A1 abundance did not increase, suggesting that the increase in UT-A1 is due, at least in part, to the decrease in the percentage of urea in the urine rather than to urine osmolality or the severity of the osmotic diuresis. In addition, it appears that the percentage of urine urea may need to decrease below a certain level because the smaller change in the percentage of urine urea induced by 2.5% NaCl diuresis did not increase UT-A1 abundance, whereas the larger change induced by 4% NaCl diuresis did increase it.
Despite the same percentage of urea in total urinary solute, the urea-supplemented rats still had a significant reduction in urine osmolality and urine urea concentration compared with control rats due to the increase in urine volume. This suggests that even though the percentage of urea in total urinary solute is the same, it is harder to achieve the same urine osmolality if the amount of total urinary solute is increased. In fact, there was a tendency for UT-A1 to increase along with the increase in total urinary solute (Fig. 6), presumably due to the simultaneous decrease in urine urea concentration and increased urinary flow rate. We speculate that UT-A1 may increase further if total urinary solute increased more than occurred in this study.
Another finding in the present study is that UT-A1 increases in the inner medullary base before it increases in the tip. In both diabetes mellitus (10) and 4% NaCl diuresis (present study), UT-A1 increases after 5 days in the inner medullary base but not in the tip. At 10–15 days, UT-A1 is increased in both the inner medullary base and tip in diabetes mellitus (10) and 4% NaCl diuresis (present study). Although we do not have an explanation for this difference in response, it may be related to the difference in the cell types present in the collecting duct in the inner medullary base vs. tip: the base contains primarily principal cells, whereas the tip contains primarily inner medullary collecting duct cells (4, 5).
To further characterize the role of urea, we asked whether UT-A1 increases in response to a low urine urea concentration or osmolality. Usually, low urine urea concentrations and low urine osmolalities occur simultaneously, making it difficult to know which is responsible for an increase in UT-A1 abundance. However, these two conditions can be dissociated if urea is used to increase the urea level in total urinary solute above that found in control rats. Urine urea concentration increased significantly in 5% urea diuresis (the increase was not statistically significant in 20% urea diuresis), whereas urine osmolality decreased significantly in 20% urea diuresis (the decrease was not statistically significant in 5% urea diuresis). Urea diuresis did not change UT-A1 abundance despite the decrease in urine osmolality and the presence of osmotic diuresis, consistent with our previous finding (14), suggesting that UT-A1 increases in response to a reduced percentage of urea in urine and/or a reduced urine urea concentration, rather than to low urine osmolality.
UT-A2 and UT-B. UT-A2 and UT-B mediate urea recycling through the thin descending limb and descending vasa recta, respectively (reviewed in Refs. 17 and 21). UT-A2 is upregulated in UT-B knockout mice, suggesting that loss of one urea-recycling pathway can be partially compensated for by increasing the other pathway (15). UT-A2 protein and mRNA abundances increase in dehydrated Sprague-Dawley rats (1, 25) or dDAVP-infused Brattleboro rats (24, 31), conditions where urine urea concentration is increased. UT-B mRNA abundance increases in vasopressin- or dDAVP-infused Brattleboro rats (20), but UT-B protein abundance decreases (30). In the present study, UT-A2 and UT-B protein abundances increase in both 5 and 20% urea diuresis, where the urine urea concentration increased, but was unchanged or decreased in NaCl diuresis and diabetes mellitus-induced glucose diuresis, conditions where the urine urea concentration is low. In addition, the renal medullary tissue urea concentration is high when the urine urea concentration is high. This suggests that UT-A2 and UT-B abundances increase when the medullary interstitial urea concentration is high, which would tend to increase intrarenal urea recycling during antidiuresis, thereby preventing the loss of urea from the medulla and maintaining medullary interstitial osmolality.
NKCC2/BSC1. NKCC2/BSC1 abundance is regulated by vasopressin-dependent and vasopressin-independent factors (reviewed in Refs. 8 and 16). Osmotic diuresis reduces sodium reabsorption in the proximal tubule, resulting in an increase in sodium delivery to the thick ascending limb. In the present study, NKCC2/BSC1 abundance increased in response to diabetes and 4% NaCl, both with and without urea, and to 20% urea. The increase in NKCC2/BSC1 is consistent with previous studies showing that NKCC2/BSC1 abundance increases when sodium delivery to the thick ascending limb increases (7, 12). NKCC2/BSC1 did not increase in response to the lesser degree of osmotic diuresis induced by 2.5% NaCl or 5% urea, suggesting that these regimens may not increase sodium delivery sufficiently to cause a measurable increase in NKCC2/BSC1.
Summary and perspective. During osmotic diuresis 1) UT-A1 abundance increases when the percentage of urea in total urinary solute and/or urine urea concentration is low, which will tend to promote the delivery of urea from the inner medullary collecting duct lumen to the inner medullary interstitium; and 2) UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high, which will tend to enhance intrarenal urea recycling and prevent the escape of urea from the renal medulla. The changes in urea transporter abundances are relatively specific because NKCC2/BSC1 abundance increases during osmotic diuresis, regardless of urea's contribution to urinary solute.
These changes also raise the possibility that urea transporters may be responding to, or sensing, urea. Is this feasible Several urea-specific signaling pathways have been identified in cultured mIMCD3 cells and the renal medulla (reviewed in Refs. 3 and 28). Thus we speculate that cells in the renal medulla may be able to sense and respond to changes in urea, perhaps by activating urea-specific signaling pathways and regulating urea transporter abundance. Future studies will be needed to test this hypothesis.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-41707, R01-DK-63657, and R01-DK-62081.
ACKNOWLEDGMENTS
Portions of this work have been published in abstract form (J Am Soc Nephrol. 14: 74A–75A, 2003) and presented at the Renal Week 2003 Meeting, San Diego, CA, November 14–17, 2003.
FOOTNOTES
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.
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Department of Pediatrics, The Catholic University of Korea, Seoul, Republic of Korea
ABSTRACT
Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.
sodium-potassium-2 chloride cotransporter; diabetes mellitus
THE RENAL MEDULLA IS THE LOCATION in which water excretion is controlled through the production of concentrated or dilute urine. Several solute transport proteins play a major role in the urinary concentrating mechanism, including urea transporters and the Na+-K+-2Cl– cotransporter (NKCC2/BSC1). Among the urea transporters, UT-A1 is expressed in the inner medullary collecting duct and is important for vasopressin-regulated urea reabsorption (reviewed in Ref. 21). UT-A2 and UT-B are expressed in the thin descending limb and descending vasa recta, respectively, and are important for intrarenal urea recycling (reviewed in Ref. 21). NKCC2/BSC1 is expressed in the thick ascending limb of the loop of Henle and is responsible for the active reabsorption of NaCl that drives the single effect to concentrate urine (reviewed in Ref. 22).
Several studies show that UT-A1, as well as NKCC2/BSC1 and aquaporin-2 (AQP2), protein abundances increase after 5 days of uncontrolled diabetes mellitus due to streptozotocin (2, 10, 11, 19, 27). We showed that UT-A1 protein abundance does not change in streptozotocin-treated Brattleboro rats, indicating that vasopressin is necessary for the increase in UT-A1 protein abundance because Brattleboro rats lack vasopressin (11). When we administered vasopressin to Brattleboro rats and then induced diabetes mellitus, UT-A1 abundance increased. Interestingly, UT-A1 abundance was increased more in vasopressin-infused diabetic Brattleboro rats than in Brattleboro rats receiving vasopressin alone. These findings imply that vasopressin is necessary but cannot be the signal to increase UT-A1 abundance because the two groups of Brattleboro rats received the same amount of vasopressin (11).
Urea is the major urinary solute in mammals, comprising 40–50% of total urinary solute in rats on a normal diet. Rats with streptozotocin-induced diabetes mellitus do have an increase in absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased (to 20%) due to the marked increase in glucose excretion (10). The ongoing osmotic diuresis due to nonreabsorbable glucose causes water to be retained in the tubule lumen, which dilutes the urine urea concentration. Several urea-specific signaling pathways have been identified in cultured mIMCD3 cells and the renal medulla (reviewed in Refs. 3 and 28), suggesting the possibility that changes in the percentage or concentration of urea in the urine, tubular fluid, or medullary interstitium could be a factor that regulates UT-A1 protein abundance. Consistent with this possibility, urea has been shown to inhibit Na+-K+-2Cl– cotransport in medullary thick ascending limb cells (9). In the present study, we tested the hypothesis that an increase in any urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus-induced glucose diuresis, whereas an increase in total urinary solute due to urea itself would not result in an increase in UT-A1. If this were true, then it would suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability. To test our hypothesis, we measured the protein abundance of UT-A1 during several models of osmotic diuresis in which the percentage of urea in total urinary solute was varied. We also measured the abundances of UT-A2, UT-B, and NKCC2/BSC1 to evaluate the specificity of any changes in UT-A1.
METHODS
Animal preparation. All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), weighing 125–200 g, received free access to a standard powdered rat chow (Testdiet 5001, Purina) containing 23% protein and 1.05% NaCl (standard diet). All rats received free access to water throughout the study. Osmotic diuresis due to glucose, NaCl, and/or urea was induced as follows.
To induce glucose diuresis, rats were made diabetic by injecting streptozotocin (60 mg/kg body wt prepared fresh in 0.1 M citrate buffer, pH 4.0, Sigma, St. Louis, MO) into a tail vein at 7 AM (10, 11, 13). Diabetes mellitus was confirmed by measuring a spot urine glucose at 24 and 48 h after streptozotocin injection (Ames N-Multistix SG, Miles, Elkhart, IN). Diabetic rats were fed the standard diet throughout the study. An additional group of rats was made diabetic, and 10% urea was added to the standard powdered diet to keep the percentage of urea in total urinary solute at the control level.
To induce NaCl diuresis, rats were fed a diet containing a total of 4 or 2.5% NaCl by adding 3 or 1.5% NaCl, respectively, to the standard powdered diet (which contains 1.05% NaCl). An additional group of rats was fed 4% NaCl and 3% urea to keep the percentage of urea in total urinary solute at the control level. NaCl was chosen as the osmolyte in this study because introducing mannitol or similar osmolytes that are not absorbed by the gastrointestinal tract would result in osmotic diarrhea rather than osmotic diuresis. In addition, it is difficult to deliver sufficient amounts of these sugars parenterally to approximate the glucose levels that are produced in diabetes mellitus, whereas NaCl can readily be delivered and appropriate levels achieved.
To induce urea diuresis, rats were fed the standard diet to which 5 or 20% urea was added. Each osmotic diuresis group (n = 6 rats) was compared with its own control group (n = 4–6 rats), which was studied in parallel. Because our previous studies showed that UT-A1 protein abundance increases consistently from 10–20 days after streptozotocin-induced diabetes mellitus (10, 11), osmotic diuresis was induced for 15–20 days in all protocols, except that 4% NaCl diuresis was induced for both 5 and 15 days to distinguish whether changes seen at 15 days were temporally distinct as they are in diabetes mellitus. Two days before death, rats were put into metabolic cages and a 24-h urine collection was obtained to measure urine volume, osmolality (model 5500 vapor pressure osmometer, Wescor, Logan, UT), and urea concentration (Infinity BUN reagent, Sigma). After 15–20 days of osmotic diuresis, rats were killed and trunk blood was collected to measure urea nitrogen (BUN) and osmolality. Blood and urine glucose concentrations were measured only in the streptozotocin-treated rats (One Touch Profile Diabetes Tracking Kit, Lifescan, Milpitas, CA). The absence of glucosuria in the other groups was confirmed using urine dip-sticks. Osmolar clearance (Cosm) and solute free water reabsorption (SFWR) were calculated as follows: Cosm = Uosm/Posm x Uvol; SFWR = Cosm – Uvol, where Uosm is urine osmolality, Posm is plasma osmolality, and Uvol is urine volume.
Tissue urea concentration. Kidneys were removed, and the medulla was dissected into outer medulla, inner medullary base, and inner medullary tip as previously described (10, 11). These tissue pieces from one kidney per rat were weighed and processed to determine interstitial urea concentration essentially as described by Schmidt-Nielsen and colleagues (23). Briefly, tissue (10 mg/piece) was weighed wet, dried over dessicant overnight, reweighed, and then urea was extracted from the dried tissue into an aqueous solution that was assayed for urea nitrogen (ThermoTrace, Melbourne, Australia). The remainder of the extract was dried under vacuum, rehydrated in a small volume, and the osmolality was determined. The contralateral kidney was processed for Western blot analysis.
Western blot analysis. Kidney tissue was placed into ice-cold isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6, 1 μg/ml leupeptin, and 2 mg/ml PMSF), homogenized; then, SDS was added to a final concentration of 1% for Western blot analysis of the total cell lysate (10, 11). Total protein in each sample was measured by a modified Lowry method (Bio-Rad DC protein assay reagent, Bio-Rad, Richmond, CA). Proteins (10 μg/lane) were size separated by SDS-PAGE using 7.5, 10, or 15% polyacrylamide gels, blotted to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI), and Western blotting was performed as described previously (10, 11).
Western blots were probed with antibodies (diluted in TBS/Tween) to 1) the UT-A1 and UT-A2 urea transporters (10, 11, 18); 2) the UT-B urea transporter (29); and 3) NKCC2/BSC1 (6, 10–12) and visualized with enhanced chemiluminescence (Amersham, Arlington Heights, IL). Blots were quantified using an imaging densitometer (GS670, Bio-Rad) and Molecular Analyst software (Bio-Rad). In all cases, parallel gels were stained with Coomassie blue to confirm uniformity of loading (data not shown). Results are expressed as arbitrary units per microgram protein.
Statistics. Data are presented as means ± SD (n), where n indicates the number of rats studied. To test for statistically significant differences between two groups, an unpaired Student's t-test was used. To test for statistically significant differences among three or more groups, an analysis of variance was used followed by a multiple-comparison, protected t-test (26).
RESULTS
Physiological parameters. Table 1 shows the urine osmolality, volume, urea, glucose, and other solutes, and plasma urea, glucose, and osmolality. Total urinary solute and urine volume were highest in rats with diabetes mellitus (glucose diuresis) and diabetes mellitus+urea and also increased in the 20% urea diuretic rats (Fig. 1). Urine osmolality was lowest in the two diabetic groups, but also reduced in the 4% NaCl+urea and the 20% urea diuretic rats. Urine urea excretion was highest in diabetes+urea and 20% urea diuretic rats but also increased in diabetic rats.
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Urine urea concentration and the relative amount (percentage) of urea in total urinary solute were decreased in diabetes mellitus and NaCl diuresis and increased in urea diuresis. The percentage of urea in total urinary solute was maintained at the control level in diabetes+urea and in the 4% NaCl+urea diuretic rats, although the urine urea concentration was reduced in these groups compared with control. However, the urine urea concentrations in rats with diabetes+urea and 4% NaCl+urea diuresis were significantly increased compared with rats with diabetes and 4% NaCl diuresis without urea supplementation, respectively.
Effect of diabetes mellitus and NaCl diuresis on UT-A1, UT-A2, UT-B, and NKCC2/BSC1 protein abundances. UT-A1 abundance in both portions of the inner medulla was significantly increased in diabetic rats (255% of control in inner medullary base, 171% of control in inner medullary tip), consistent with our previous results (10, 11), and in rats with 4% NaCl diuresis for 15 days (166% of control in inner medullary base, 162% of control in inner medullary tip) (Fig. 2). It was unchanged in 2.5% NaCl diuretic rats. Because UT-A1 abundance varies with time after streptozotocin injection (10), we tested whether there was a temporal change in UT-A1 after induction of 4% NaCl diuresis. UT-A1 abundance increased at 5 days of 4% NaCl diuresis in the inner medullary base, but not in the inner medullary tip, similar to the pattern of changes in UT-A1 at 5 days of diabetes (10).
In contrast, UT-A2 abundance in the outer medulla was significantly decreased in diabetic rats (66% of control) and in rats with 2.5% NaCl diuresis (72% of control) but was unchanged in 4% NaCl diuresis (Fig. 3) rats. UT-B abundance was unchanged in any portion of the medulla with either diabetes or 4% NaCl diuresis (Fig. 4). NKCC2/BSC1 abundance in the outer medulla (Fig. 5) was significantly increased in both diabetic rats (143% of control) and 4% NaCl diuretic (130% of control) but not in 2.5% NaCl diuretic rats.
Effect of urea feeding on diabetic and 4% NaCl diuretic rats. The data in Figs. 1 and 2 suggest the hypothesis that a decrease in urine urea below a certain level may be a signal that increases UT-A1 abundance in the inner medulla. To test this hypothesis, diabetic and 4% NaCl diuretic rats were fed supplemental urea to restore the percentage of urea in urine solute to control levels (Table 1, Fig. 1). Diabetic rats and 4% NaCl diuretic rats fed urea supplements did not show significant differences in UT-A1 (Fig. 6), UT-A2 (Fig. 7), or UT-B abundances (Fig. 8) compared with control rats, suggesting that restoring the percentage of urea in total urinary solute reverses the diabetes- or 4% NaCl diuresis-induced upregulation of UT-A1 abundance. NKCC2/BSC1 abundance (Fig. 9) was significantly increased in both rats with diabetes+10% urea diuresis (124% of control) and 4% NaCl+3% urea diuresis (121% of control). However, the percentage increases were somewhat smaller than the increases in rats with diabetes or 4% NaCl diuresis alone.
Effect of urea diuresis. UT-A1 abundance was not changed in rats with 5 or 20% urea diuresis in either the inner medullary base or tip (Fig. 6), even though these rats had severe osmotic diuresis. Considering that the urine urea concentration is higher, but urine osmolality is lower, than those of control rats (Table 1, Fig. 1), this suggests that UT-A1 is responding to the urine urea concentration, rather than urine osmolality. In contrast, UT-A2 abundance in the outer medulla was significantly increased with both the 5% urea (163% of control) and 20% urea (155% of control) diuresis (Fig. 7). UT-B abundance in the outer medulla was also significantly increased in both rats with 5% urea (150% of control) and 20% urea (130% of control) diuresis but was unchanged in the inner medullary base (Fig. 8). NKCC2/BSC1 abundance showed a tendency to increase in both rats with 5 and 20% urea diuresis, but the increase was statistically significant only in 20% urea diuretic rats (Fig. 9).
Tissue urea concentration. To determine whether the changes in urine urea reflected tissue urea, we determined tissue urea concentrations in 20% urea diuretic and 4% NaCl diuretic rats. Compared with control rats, tissue urea levels were significantly increased in all medullary regions in the 20% urea diuretic rats (Table 2). There was no difference in the tissue urea content of the 4% NaCl diuretic rats compared with control rats.
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DISCUSSION
UT-A1. The major finding in this study is that UT-A1 protein abundance increases during osmotic diuresis whenever the relative amount (percentage) of urea in total urinary solute decreases. Regardless of whether glucose (diabetes mellitus) or NaCl induced the osmotic diuresis, thereby reducing the percentage of urea in the urine, UT-A1 abundance increased. When osmotic diuresis was induced by urea, the percentage of urea in the urine did not decrease and UT-A1 abundance did not increase.
To further test the role of urine urea, rats undergoing osmotic diuresis due to glucose or NaCl were given urea supplements to prevent the decrease in the percentage of urea in the urine. With the urea-supplemented diets, the percentage of urea in total urinary solute was adjusted so that is was similar to that in control rats. In both of the urea-supplemented groups, UT-A1 abundance did not increase, suggesting that the increase in UT-A1 is due, at least in part, to the decrease in the percentage of urea in the urine rather than to urine osmolality or the severity of the osmotic diuresis. In addition, it appears that the percentage of urine urea may need to decrease below a certain level because the smaller change in the percentage of urine urea induced by 2.5% NaCl diuresis did not increase UT-A1 abundance, whereas the larger change induced by 4% NaCl diuresis did increase it.
Despite the same percentage of urea in total urinary solute, the urea-supplemented rats still had a significant reduction in urine osmolality and urine urea concentration compared with control rats due to the increase in urine volume. This suggests that even though the percentage of urea in total urinary solute is the same, it is harder to achieve the same urine osmolality if the amount of total urinary solute is increased. In fact, there was a tendency for UT-A1 to increase along with the increase in total urinary solute (Fig. 6), presumably due to the simultaneous decrease in urine urea concentration and increased urinary flow rate. We speculate that UT-A1 may increase further if total urinary solute increased more than occurred in this study.
Another finding in the present study is that UT-A1 increases in the inner medullary base before it increases in the tip. In both diabetes mellitus (10) and 4% NaCl diuresis (present study), UT-A1 increases after 5 days in the inner medullary base but not in the tip. At 10–15 days, UT-A1 is increased in both the inner medullary base and tip in diabetes mellitus (10) and 4% NaCl diuresis (present study). Although we do not have an explanation for this difference in response, it may be related to the difference in the cell types present in the collecting duct in the inner medullary base vs. tip: the base contains primarily principal cells, whereas the tip contains primarily inner medullary collecting duct cells (4, 5).
To further characterize the role of urea, we asked whether UT-A1 increases in response to a low urine urea concentration or osmolality. Usually, low urine urea concentrations and low urine osmolalities occur simultaneously, making it difficult to know which is responsible for an increase in UT-A1 abundance. However, these two conditions can be dissociated if urea is used to increase the urea level in total urinary solute above that found in control rats. Urine urea concentration increased significantly in 5% urea diuresis (the increase was not statistically significant in 20% urea diuresis), whereas urine osmolality decreased significantly in 20% urea diuresis (the decrease was not statistically significant in 5% urea diuresis). Urea diuresis did not change UT-A1 abundance despite the decrease in urine osmolality and the presence of osmotic diuresis, consistent with our previous finding (14), suggesting that UT-A1 increases in response to a reduced percentage of urea in urine and/or a reduced urine urea concentration, rather than to low urine osmolality.
UT-A2 and UT-B. UT-A2 and UT-B mediate urea recycling through the thin descending limb and descending vasa recta, respectively (reviewed in Refs. 17 and 21). UT-A2 is upregulated in UT-B knockout mice, suggesting that loss of one urea-recycling pathway can be partially compensated for by increasing the other pathway (15). UT-A2 protein and mRNA abundances increase in dehydrated Sprague-Dawley rats (1, 25) or dDAVP-infused Brattleboro rats (24, 31), conditions where urine urea concentration is increased. UT-B mRNA abundance increases in vasopressin- or dDAVP-infused Brattleboro rats (20), but UT-B protein abundance decreases (30). In the present study, UT-A2 and UT-B protein abundances increase in both 5 and 20% urea diuresis, where the urine urea concentration increased, but was unchanged or decreased in NaCl diuresis and diabetes mellitus-induced glucose diuresis, conditions where the urine urea concentration is low. In addition, the renal medullary tissue urea concentration is high when the urine urea concentration is high. This suggests that UT-A2 and UT-B abundances increase when the medullary interstitial urea concentration is high, which would tend to increase intrarenal urea recycling during antidiuresis, thereby preventing the loss of urea from the medulla and maintaining medullary interstitial osmolality.
NKCC2/BSC1. NKCC2/BSC1 abundance is regulated by vasopressin-dependent and vasopressin-independent factors (reviewed in Refs. 8 and 16). Osmotic diuresis reduces sodium reabsorption in the proximal tubule, resulting in an increase in sodium delivery to the thick ascending limb. In the present study, NKCC2/BSC1 abundance increased in response to diabetes and 4% NaCl, both with and without urea, and to 20% urea. The increase in NKCC2/BSC1 is consistent with previous studies showing that NKCC2/BSC1 abundance increases when sodium delivery to the thick ascending limb increases (7, 12). NKCC2/BSC1 did not increase in response to the lesser degree of osmotic diuresis induced by 2.5% NaCl or 5% urea, suggesting that these regimens may not increase sodium delivery sufficiently to cause a measurable increase in NKCC2/BSC1.
Summary and perspective. During osmotic diuresis 1) UT-A1 abundance increases when the percentage of urea in total urinary solute and/or urine urea concentration is low, which will tend to promote the delivery of urea from the inner medullary collecting duct lumen to the inner medullary interstitium; and 2) UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high, which will tend to enhance intrarenal urea recycling and prevent the escape of urea from the renal medulla. The changes in urea transporter abundances are relatively specific because NKCC2/BSC1 abundance increases during osmotic diuresis, regardless of urea's contribution to urinary solute.
These changes also raise the possibility that urea transporters may be responding to, or sensing, urea. Is this feasible Several urea-specific signaling pathways have been identified in cultured mIMCD3 cells and the renal medulla (reviewed in Refs. 3 and 28). Thus we speculate that cells in the renal medulla may be able to sense and respond to changes in urea, perhaps by activating urea-specific signaling pathways and regulating urea transporter abundance. Future studies will be needed to test this hypothesis.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-41707, R01-DK-63657, and R01-DK-62081.
ACKNOWLEDGMENTS
Portions of this work have been published in abstract form (J Am Soc Nephrol. 14: 74A–75A, 2003) and presented at the Renal Week 2003 Meeting, San Diego, CA, November 14–17, 2003.
FOOTNOTES
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.
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