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Am J Physiol Regul Integr Comp Physiol 285: R1355-R1365, 2003. First published August 21, 2003; doi:10.1152/ajpregu.00207.2003
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THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS

Urea transporter expression in aging kidney and brain during dehydration

M.-M. Trinh-Trang-Tan,1,2 G. Geelen,3 L. Teillet,4 and B. Corman2

1Institut National de la Santé et de la Recherche Médicale U76, Institut National de Transfusion Sanguine, F-75015 Paris; 2Service de Biologie Cellulaire, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex; 3Laboratoire de Physiologie de l'Environnement, Université Claude Bernard, Faculté de Médecine Grange-Blanche, F-69373 Lyon Cedex 8; and 4Assistance Publique-Hôpitaux de Paris, Hôpital Sainte-Perrine, F-75781 Paris, France

Submitted 17 April 2003 ; accepted in final form 14 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aging is commonly associated with defective urine-concentrating ability. The present study examined how the kidney and the brain of senescent (30-mo-old) female WAG/Rij rats respond to dehydration induced by 2 days of water deprivation in terms of urea transporter (UT) regulation. In euhydrated situation, senescent rats exhibited similar vasopressin plasma level but lower urine osmolality and papillary urea concentration and markedly reduced kidney UT-A1, UT-A3, and UT-B1 abundances compared with adult (10-mo-old) rats. Senescent rats responded to dehydration similarly to adult rats by a sixfold increase in vasopressin plasma level. Their papillary urea concentration was doubled, without, however, attaining that of dehydrated adult rats. Such an enhanced papillary urea sequestration occurred with a great fall of both UT-A1 and UT-A3 abundances in the tip of inner medulla and an increased UT-A1 abundance in the base of inner medulla. UT-A2 and UT-B1 were unchanged. These data suggest that the inability of control and thirsted senescent rats to concentrate urine as much as their younger counterparts derives from lower papillary urea concentration. In aging brain, UT-B1 abundance was increased twofold together with a fourfold increase in aquaporin-4 abundance. Dehydration did not alter the abundance of these transporters.

papillary osmolality; urea transporter-A1, -A2, -A3, and -B1; aquaporin-4; vasopressin

UREA TRANSPORTERS (UTs) have been cloned during the past decade, and their functional relevance in several organs is under investigation. UT-A1, -A2, -A3, and -A4 are encoded by a same gene (14, 28) and expressed exclusively in the kidney (17). UT-B1 is encoded by a different gene (33, 34, 48) and is expressed in a number of tissues, including erythrocytes, kidney, brain, and testis (32, 45, 46). In the kidney, these UTs are located in the medulla, where they participate in the urinary-concentrating process (38, 47). Urea medullary accumulation is optimized by UT-A1 and UT-A3, which deliver urea from the lumen of terminal collecting ducts into medulla interstitium, and UT-A2 and UT-B1 present in descending thin limbs and descending vasa recta, respectively, which recycle urea and minimize its escape from the medulla. The corticopapillary osmotic gradient involving urea and NaCl accumulation drives the aquaporin (AQP)-mediated water reabsorption, under vasopressin (AVP) control, from the lumen of collecting ducts.

In the brain, UT-B1 protein was found in astrocyte end feet processes surrounding vascular microvessels and in ependymal cells lining ventricles (46). Such a location implies a role in brain parenchyme clearance of urea in excess, potentially toxic at high concentration (7). In erythrocytes, UT-B1 is believed to protect the cells from excess shrinking and swelling when passing through the hypertonic inner medulla (IM) (21).

In humans and rodents, urine-concentrating ability declines with age, resulting in polyuria and impaired renal concentrating ability (5, 6, 10, 12, 24). Such a polyuria associated with insufficient water intake due to abnormal thirst thresholds frequently results in dehydration in elderly subjects (39, 42). We have previously observed that senescent female WAG/Rij rats, which do not exhibit age-associated chronic progressive nephropathy and pituitary tumors, are polyuric despite normal AVP secretion and plasma concentrations under normal hydration (16). Impaired expression of AQP-2 and -3 in kidney medulla appears to be an important cause of age-related polyuria (37). In addition, we have recently observed that UT-A1 and UT-B1 are also greatly underexpressed in these aging rats (9).

In the current study, we analyzed in the same rat model, the adaptation of aging kidney and brain to dehydration, with emphasis on the possible involvement of urea and its transporters. Maximal urine-concentrating ability, plasma AVP levels, and kidney medulla urea content were determined. The changes in UT expressions were evaluated in kidney and brain in adult and senescent animals under control condition and after dehydration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and water deprivation protocol. Female WAG/Rij rats were born, raised, and maintained in the animal care facility of CEA/Saclay (Gif-sur-Yvette) on a 12:12-h light-dark cycle, with 50% ambient humidity and 21°C temperature. The suitability of the WAG/Rij rats for the study of kidney aging is related to the absence of chronic progressive glomerulosclerosis with age (4, 10). Senescent 30-mo-old rats were compared with adult 10-mo-old rats. These animals are known not to increase their body weight by more than 10-20% after they have reached adulthood.

Metabolic cage experiments. Twelve 10-mo-old and twelve 30-mo-old rats (body wt 195 ± 4 and 220 ± 5 g, respectively) were housed in metabolic cages for 2 days of habituation followed by a 2-day control period with water intake and urine flow rate determination. Urine was collected under mineral oil to minimize evaporation. Urine osmolality was determined with a Roebling Automatik osmometer. The drinking water was then removed for 2 days for half of the rats in each age group. Rats lost ~10% of body weight after the thirsting period.

Measurement of plasma AVP. Animals were killed by decapitation at the end of the 2-day dehydration period, and trunk blood was rapidly collected and centrifuged at 4°C. Plasma AVP concentration was measured by RIA with antiserum K9-IV (19) (gift of Dr. L. C. Keil, NASA Ames Research Center) and 125I-labeled iodo-AVP (16, 19). The minimum sensitivity of the assay was 0.25 pg/assay. For each animal, determination of plasma AVP concentration was performed in triplicate with independent standard curves.

Measurement of papillary osmolality and urea concentration. Kidneys were rapidly removed, and the whole papilla was excised on ice from each kidney, weighed, and thoroughly homogenized with a motor-driven homogenizer, after addition of 600 µl of distilled water. Homogenate osmolality was measured with a Roebling Automatik osmometer, and urea concentration was measured using a specific kit (Bio-Mérieux, Lyon, France). Papilla osmolality and urea content were calculated assuming that 80% of papilla wet weight is water (2).

Antibodies. UT-A1 and UT-A2 were revealed using an affinity-purified rabbit polyclonal antibody raised against the COOH-terminal peptide sequence common to rat UT-A1 and UT-A2 (Alpha Diagnostics International, San Antonio, TX). Affinity-purified UT-A3 polyclonal antibody (Q-2695-2) was kindly provided by Dr M. A. Knepper and previously characterized (44). UT-B was detected by an affinity-purified rabbit polyclonal antibody against the COOH terminus of rat UT-B1 (46). AQP-4 was detected using an affinity-purified rabbit polyclonal antibody raised against the COOH-terminal peptide sequence of rat AQP-4 (Alpha Diagnostics International). Glial fibrillary acid protein (GFAP) was revealed with a polyclonal antibody raised in rabbits against bovine GFAP and purified using protein A (Promega, Madison, WI).

Western blot analysis. Because UTs are not evenly expressed in kidney inner stripe of outer medulla (ISOM) and in the IM, the ISOM was divided into upper and lower halves, and the IM was divided into base (upper third) and tip (two lower thirds). Given the difference in cell type composition of the various cerebrum areas, brain coronal sections were collected from the same midcerebrum areas for each rat. The tissue fragments were thoroughly homogenized in ice-cold lysis buffer (250 mmol/l sucrose, 10 mmol/l triethanolamine, pH 7.6) containing protease inhibitors (Complete Mini EDTA-free protease inhibitor cocktail tablets, Roche Diagnostics, Mannheim, Germany). Homogenate protein concentration was determined by the Bradford method (Bio-Rad, Hercules, CA). Samples were then solubilized in Laemmli buffer and heated at 65°C for 10 min before loading. Fifteen or thirty micrograms of protein were separated by SDS-PAGE (10%) and transferred to polyvinylidene difluoride (PVDF) membranes (NEN, Boston, MA). Blots were blocked for 45 min at room temperature with PBS containing 5% nonfat dry milk, followed by incubation with the primary antibody for 2 h at room temperature. The membranes were then thoroughly washed and incubated for 60 min with a goat peroxidase-conjugated anti-rabbit IgG polyclonal antibody. Bands were visualized on Hyperfilm-ECL (Amersham) by chemiluminescence (ECL+, NEN). Equal protein loading was verified by Coomassie blue staining of the PVDF membranes at the end of the experiment. After scanning, semi-quantitative densitometry was performed on films using NIH Image software. A background value was subtracted from the density value of each band. The abundance of each transporter, for each rat, was determined by combining the densities of unglycosylated and/or glycosylated forms.

Statistics. Results were expressed as means ± SE. Differences between control and thirsted rats at both ages were analyzed by one-way ANOVA. Differences were considered significant for P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Data from metabolic cage experiments are summarized in Table 1. In line with previous reports (9, 16, 37), the urine output normalized to body weight in senescent rats was significantly higher and urine osmolality was lower than in adult rats under control condition (water ad libitum). At both ages, drinking water withdrawal for 2 days reduced urine output by 85-90%. However, senescent rats still exhibited higher urine output and lower urine osmolality than adult rats during thirsting.


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  		Table 1. In vivo parameters of 10- and 30-mo-old female WAG/Rij rats in control condition and after 2 days of water deprivation

 

AVP plasma concentration. The AVP plasma level of senescent rats was not significantly different from that of adult rats (1.7 ± 0.2 and 1.4 ± 0.2 pg/ml, respectively, n = 6). Thirsting for 2 days caused a similar sixfold increase of plasma AVP concentration at both ages, resulting in the same elevated hormone concentrations (10.0 ± 1.0 and 10.9 ± 0.6 pg/ml, respectively, n = 6).

Papillary osmolality and urea content. Kidney and papilla weights were unaltered by 2-day water deprivation (data not shown). As can be seen in Table 1, papillary osmolalities were about half of urine osmolalities. Indeed, total papillary osmolality involves the osmotic gradient of whole IM, whereas urine osmolality reflects that of the very tip of papilla. Water deprivation significantly and equally incremented papillary osmolality (+350 mosmol/kgH2O) and urea concentration (+200 mmol/l) at both ages (Table 1). It can be pointed out that papilla osmolality and urea concentration remained significantly lower in dehydrated 30-mo-old compared with dehydrated 10-mo-old animals, because of lower predehydration values. As calculated from Table 1, in control 30- and 10-mo-old rats, urea accounted for 17 and 25% of papilla total osmoles, respectively. This proportion was increased to 31 and 34%, respectively, after water deprivation, indicating an increased contribution of urea to medulla hypertonicity.

UT-A1, UT-A2, UT-A3, and UT-B1 expression in kidney medulla. UT-A1 proteins in control 10-mo-old rats were predominantly expressed in IM tip [terminal inner medullary collecting ducts (IMCDs)] as 97- and 117-kDa glycosylated forms (Fig. 1). In control 30-mo-old rats, UT-A1 protein expression in IM tip was very low (Fig. 1A). Water deprivation caused a striking fall in the abundance of both forms of UT-A1 in 10-mo-old rats (Fig. 1B). In 30-mo-old rats, dehydration did not make UT-A1 detectable in the tip of IM, even with higher amounts of protein (data not shown). In line with previous observations, UT-A1 was hardly detected in IM base (initial IMCDs) of rat kidney due to very weak expression. Loading high protein amount and prolonged film exposure duration revealed that thirsting induced a significant increase in the 117-kDa form of UT-A1 in both 10- and 30-mo-old rats (Fig. 2).



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  		Fig. 1. Immunoblots comparing the expression of urea transporter (UT)-A1 proteins (97- and 117-kDa isoforms) in the tip of inner medulla (IM) of control (Cont) and water-deprived (WD) 10- and 30-mo-old rats. A: blots were probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence common to rat UT-A1 and UT-A2 (0.25 µg/ml). Each lane, representing an individual rat, was loaded with 15 µg protein. B: density quantification of combined isoforms revealed that UT-A1 was massively underexpressed in control 30-mo-old compared with control 10-mo-old rats. It also disclosed a lowering effect of water deprivation in 10-mo-old rats. In 30-mo-old rats, dehydration did not make UT-A1 detectable even with higher amounts of protein (data not shown). *P < 0.05 vs. respective control condition.

 


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  		Fig. 2. Immunoblots comparing the expression of UT-A1 proteins (97- and 117-kDa isoforms) in the base of IM of control and water-deprived 10- and 30-mo-old rats. A: blots were probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence common to rat UT-A1 and UT-A2 (0.25 µg/ml). Each lane, representing an individual rat, was loaded with 30 µg protein. B: density quantification of combined isoforms reveals that UT-A1 is significantly increased by water deprivation at both ages. *P < 0.05 vs. respective control condition.

 

UT-A3 has been cloned recently and identified as a second urea transporter of terminal IMCD (44). It is expressed as two glycosylated bands of 44 and 67 kDa in the tip of IM (44) (Fig. 3). The third band present between these two was a nonspecific band, exhibited by WAG/Rij but not Sprague-Dawley rats, and unaltered by age and dehydration. The changes in the abundance of UT-A3 (44- and 67-kDa bands) induced by aging and dehydration were akin to those of UT-A1: UT-A3 was abated greatly by aging and significantly by dehydration, with a major disappearance of the 44-kDa protein (Fig. 3).



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  		Fig. 3. Immunoblots comparing the expression of UT-A3 proteins (44- and 67-kDa isoforms) in the tip of IM of control and water-deprived 10- and 30-mo-old rats. A: blots were probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence specific to rat UT-A3 (0.10 µg/ml). Each lane, representing an individual rat, was loaded with 15 µg protein. The band indicated by the arrowhead is a nonspecific band abated by further dilution of the antibody, unaltered by age or water deprivation, and present only in WAG/Rij strain. B: density quantification of combined isoforms reveals a drastic fall in UT-A3 at 30 mo of age (a). It also discloses a lowering effect of water deprivation at 10-mo of age (b). Prolonged film exposure time did not reveal any change in UT-A3 expression induced by WD in 30-mo-old rats. *P < 0.05 vs. respective control condition.

 

Due to its main location in the distal part of descending thin limbs (DTLs) of rat short-looped nephrons, UT-A2 is predominantly detected in the lower half of ISOM as a broad band around 50 kDa. It is hardly detected in the upper half of ISOM due to its absence in the early part of DTLs (31). The abundance of UT-A2 in these two subzones was unchanged by aging as well as by water deprivation (Figs. 4 and 5).



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  		Fig. 4. Immunoblots comparing the expression of UT-A2 proteins (broad band around 50 kDa) in the lower half of inner stripe of outer medulla (ISOM) of control and water-deprived 10- and 30-mo-old rats. A: each lane, representing an individual rat, was loaded with 15 µg protein and probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence common to rat UT-A1 and UT-A2 (0.25 µg/ml). B: density quantification reveals that UT-A2 abundance is equal in 30-mo-old and 10-mo-old rats and is unaltered by water deprivation at both ages.

 


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  		Fig. 5. Immunoblots comparing the expression of UT-A2 proteins (broad band around 50 kDa) in the upper half of ISOM of control and water-deprived 10- and 30-mo-old rats. A: each lane, representing an individual rat, was loaded with 15 µg protein and probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence common to rat UT-A1 and UT-A2 (0.25 µg/ml). B: density quantification reveals that UT-A2 abundance is equal in 30-mo-old and 10-mo-old rats and is unaltered by water deprivation at both ages.

 

In the kidney, UT-B1 is present in the endothelium of descending vasa recta irrigating the medulla. It is expressed as an unglycosylated form of 29 kDa and several glycosylated forms with 45- to 56-kDa apparent molecular masses. Under normal hydration, its abundance was significantly higher in IM tip (Fig. 6) and significantly lower in IM base (Fig. 7) of 30-mo-old rats compared with 10-mo-old rats. UT-B1 abundance was not altered by dehydration in both subregions and at both ages (Figs. 6 and 7).



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  		Fig. 6. Immunoblots comparing the expression of UT-B1 proteins (29-kDa unglycosylated and 45- to 56-kDa glycosylated forms) in the base of IM of control and in water-deprived 10- and 30-mo-old rats. A: each lane, representing an individual rat, was loaded with 15 µg protein and probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence of rat UT-B1 (0.25 µg/ml). B: density quantification reveals that UT-B1 abundance is significantly higher in control 30-mo-old than in control 10-mo-old rats and is unaltered by water deprivation at both ages. *P < 0.05 vs. respective control condition.

 


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  		Fig. 7. Immunoblots comparing the expression of UT-B1 proteins (29-kDa unglycosylated and 45- to 56-kDa glycosylated forms) in the tip of IM of control and water-deprived 10- and 30-mo-old rats. A: each lane, representing an individual rat, was loaded with 15 µg protein and probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence of rat UT-B1 (0.25 µg/ml). B: density quantification reveals that UT-B1 abundance is significantly lower in control 30-mo-old than in control 10-mo-old rats and is unaltered by water deprivation at both ages. *P < 0.05 vs. respective control condition.

 

UT-B1 and AQP-4 expression in cerebrum. In brain, UT-B1 and AQP-4 are colocalized in astrocytes and ependymal cells. UT-B1 is revealed by immunoblotting as two bands of 29 kDa (unglycosylated form) and 33 kDa (glycosylated form). It was overexpressed by twofold in 30-mo-old rats (Fig. 8). Immunoblotting of the same brain samples for AQP-4 detection revealed a fourfold higher expression of AQP-4 in the aging brain (Fig. 9). The abundance of GFAP, the usual marker of astrocytes and ependymal cells, was increased by 25% with age (data not shown), a variation much smaller than the doubling in UT-B1 or the fourfold increase in AQP-4. Thirsting had no effect on UT-B1 and AQP-4 abundance at both ages (Figs. 8 and 9).



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  		Fig. 8. Immunoblots comparing the expression of UT-B1 proteins (29-kDa unglycosylated and 33-kDa glycosylated form) in cerebrum of control and water-deprived 10- and 30-mo-old rats. A: each lane, representing an individual rat, was loaded with 15 µg protein and probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence of rat UT-B1 (0.25 µg/ml). B: density quantification reveals that UT-B1 abundance is significantly higher by >2-fold in control 30-mo-old than in control 10-mo-old rats and is unaltered by water deprivation at both ages. *P < 0.05 vs. respective control condition.

 


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  		Fig. 9. Immunoblots comparing the expression of aquaporin (AQP)-4 proteins (31-kDa unglycosylated and 52-kDa glycosylated form) in cerebrum of control and in water-deprived 10- and 30-mo-old rats. A: each lane, representing an individual rat, was loaded with 15 µg protein and probed with an affinity-purified polyclonal antibody raised against the COOH-terminal sequence of rat AQP-4 (0.25 µg/ml). B: density quantification reveals that AQP-4 abundance is significantly higher by 4-fold in control 30-mo-old than in control 10-mo-old rats and is unaltered by water deprivation at both ages. *P < 0.05 vs. respective control condition.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Four important findings are revealed in the present study conducted on the model of aging represented by female WAG/Rij rats: 1) the expression of UT-A3, akin to that of UT-A1, is massively abated in the tip of IM of euhydrated aging rats; 2) the decline in urine osmolality observed in senescent euhydrated as well as dehydrated rats pertains to lower papillary urea content; 3) the aging kidney, however, is still responsive to the challenge of prolonged water deprivation, thanks to improved urea medullary accumulation, and despite decreased expression of both UT-A1 and UT-A3 in terminal collecting duct; and 4) aging is associated with a brain overexpression of UT-B1 and AQP-4 that is not modified by thirsting.

The impaired concentrating ability of aging kidney has been well documented (6, 12, 16, 25, 40). Under conditions of normal hydration, this defect was attributed to abnormal expression of AQPs and UTs in kidney IM and to important reduction in papillary urea concentration, whereas AVP secretion was preserved (16, 37, 43). A new finding of the present study is that expression of UT-A3, the other terminal collecting duct urea transporter (17, 44), is also impaired akin to UT-A1 in senescent rats (8, 9). Hence, such a defect may be an additional cause for the age-related impaired urine-concentrating activity of euhydrated senescent rats. In the present study, senescent WAG/Rij rats evolved effective antidiuretic response to dehydration, as revealed by a doubling of urine osmolality and papillary urea content. Proper adjustment of plasma AVP levels occurred as noticed from the sixfold increase in control adult and aging rats of this study and in previous ones (16, 23, 27). However, a defect in maximal urine osmolality persisted, compared with dehydrated younger adult rats. The 35% residual deficit in urine osmolality of thirsted senescent compared with thirsted adult rats correlated with a 30% deficit in papillary urea concentration. This finding suggests that the age-related impaired urinary-concentrating ability of the aging kidney under both normal hydration and water deprivation is due to reduced urea supply to the medulla.

The building up of high medullary urea gradient implies high urea permeability in terminal IMCDs, descending thin limbs, and descending vasa recta, mediated by UT-A1 and UT-A3, UT-A2, and UT-B1, respectively. Table 2 summarizes the changes induced by aging and water deprivation for urea transporter abundance in kidney medulla subzones. The present study reveals that UT-A1 abundance is decreased in the tip and increased in the base of IM during thirsting. Such a pattern of changes may signify situations of exaggerated antidiuresis inasmuch as it was also found in rats chronically infused with dDAVP, a synthetic analog of vasopressin only endowed with antidiuretic effect (8).


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  		Table 2. Changes in UT-A1, UT-A2, UT-A3, and UT-B1 in kidney medulla subzones induced by aging and 2-day water deprivation

 

The current finding of an underexpression in IM tip is corroborated by the recent finding, obtained by immunogold method, of a striking decrease of UT-A1 immunoreactivity intensity in IMCD of Sprague-Dawley rats deprived of drinking water for 3 days (20). Because glucocorticoids were shown to downregulate UT-A1 expression in the terminal portion of IMCDs (29, 35), this change may pertain to the water deprivation-induced increase in glucocorticoid secretion (1, 13, 36). However, the decrease in UT-A1 abundance in the tip of IM is not consistent with the increased urea permeability of terminal collecting duct reported in dehydrated Sprague-Dawley rats (18). By contrast to IM tip, IM base undergoes a doubling of UT-A1 abundance during thirsting. Such an increase would mediate the higher basal urea permeability of initial IMCDs found by Kato et al. (18) in 2- to 3-day-dehydrated rats. Hence, the increased delivery of urea to IM interstitium may proceed through an increase in the number of UT-A1 proteins not in terminal but in initial IMCDs. It may be also assumed that during dehydration, UT-A1 proteins, although in low abundance, are totally phosphorylated in the presence of elevated endogenous AVP levels, thereby becoming the functional form for urea transport as proposed by Zhang and colleagues (49).

As shown here, UT-A3 expression does not compensate for the defective UT-A1 abundance in the tip of IM because it is also strongly lowered by thirsting. This finding is in accordance with the recent finding of abated UT-A3 immunoreactivity on kidney sections of dehydrated rats (20). Finally, UT-independent mechanisms may be involved in papillary urea sequestration during thirsting. It may be the drastic slowdown of urine flow rate in the lumen of terminal collecting ducts that favors passive diffusion of urea. Alternatively, IMCDs may adapt to dehydration by stabilization of apical membrane in a state of high urea permeability, as it does for osmotic water permeability, and/or by the opening up of paracellular pathways as suggested by Flamion et al. (15).

UT-A2, the UT of the distal part of thin descending limbs, and UT-B1, the UT of endothelial cells of descending vasa recta, are most likely involved in papillary urea recycling. UT-A2 expression did not differ significantly between 10- and 30-mo-old rats, in both the lower and the upper half of ISOM, and was unchanged by dehydration-induced antidiuresis. Such a result regarding protein expression differs from that regarding mRNA expression, which was found to be increased by thirsting in both rats (3) and mice (14). It also differs from antidiuresis induced by chronic dDAVP infusion, which, despite much less pronounced antidiuresis and urine osmolality, resulted in increased abundance of UT-A2 protein transporter in both regions of ISOM (8).

We have previously reported a lower abundance of UT-B1 in total IM of senescent rats compared with adult rats (9). The present study further discloses a different regional expression for UT-B1 within IM in the course of aging. It shows that senescent rats have a much lower abundance of the transporter in the base of IM, which constitutes the vast majority of total IM tissue, thereby confirming previous observations. Furthermore, it reveals that UT-B1 protein content in the tip of the IM is higher in senescent rats, perhaps thereby compensating for UT-A1 underexpression. Water deprivation did not change significantly the abundance of UT-B1 in IM, a result at variance with the decrease in UT-B1 expression caused by dDAVP-induced antidiuresis (8, 46).

To determine whether the age-related changes in UT-B1 are specific to the kidney, the expression of the transporter was examined in the brain, where the transporter was found in astrocytes and ependymal cells (7, 46). We disclosed that the brain abundance of UT-B1 protein was doubled in aged rats compared with adult rats. This increase is definitely abnormal because it is much higher than the 20-25% age-related increases in the number of astrocytes and GFAP immunoreactivity previously reported in aging rodents (11, 26) and here in WAG/Rij rats. Furthermore we also found that brain expression of AQP-4, which is colocated with UT-B1 in astrocytes and ependymal cells (30), was considerably increased in aging rats. The overexpression of AQP-4 in aging brain may favor cerebral edema because it has been shown that AQP-4 deletion reduces brain edema after acute water intox-ication and ischemic stroke (22). Future studies are needed to determine whether the age-related overexpression of UT-B1 and AQP-4 in brain results in dysregulation of cerebral volume and tonicity with subsequent alterations of brain functions, given that astrocytes are now recognized as active modulators of neuronal activity (41). Dehydration did not modify brain UT-B1 and AQP-4 abundances.

In summary, the present study reports age-related differences in UT-A1, UT-A3, and UT-B1 urea transporter abundance in the kidney. It also reveals an overexpression of UT-B1 and AQP-4 in aging brain, the possible role of which, in dysfunction of cerebral volume and tonicity, needs further study. Prolonged thirsting results in a pronounced antidiuresis favored by proper kidney medulla urea accumulation yet a downregulation of UT-A1 and UT-A3 in senescent and adult rats as well. However, during thirsting, the renal concentrating capacity of senescent rats remains lower than that of adult rats likely because of lower papillary osmolality and urea content. In contrast, UT-B1 expression in both kidney and brain is unchanged by water deprivation.


   ACKNOWLEDGMENTS
 
We are grateful to J.-C. Robillard for outstanding animal care and G. Augoyard for skill in performing AVP assays.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M.-M. Trinh-Trang-Tan, INSERM U76, Institut National de Transfusion Sanguine, 6, rue Alexandre Cabanel, F-75015 Paris, France (E-mail: trinh{at}idf.inserm.fr).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aguilera G, Lightman SL, and Kiss A. Regulation of the hypothalamic-pituitary-adrenal axis during water deprivation. Endocrinology 132: 241-248, 1993.[Abstract]
  2. Apostol E, Ecelbarger CA, Terris J, Bradford AD, Andrews P, and Knepper MA. Reduced renal medullary water channel expression in puromycin aminonucleoside-induced nephrotic syndrome. J Am Soc Nephrol 8: 15-24, 1997.[Abstract]
  3. Bagnasco SM, Peng T, Nakayama Y, and Sands JM. Differential expression of individual UT-A urea transporter isoforms in rat kidney. J Am Soc Nephrol 11: 1980-1986, 2000.[Abstract/Free Full Text]
  4. Baylis C and Corman B. The aging kidney: insights from experimental studies. J Am Soc Nephrol 9: 699-709, 1998.[Abstract]
  5. Beck N and Yu BP. Effect of aging on urinary concentrating mechanism and vasopressin-dependent cAMP in rats. Am J Physiol Renal Fluid Electrolyte Physiol 243: F121-F125, 1982.[Abstract/Free Full Text]
  6. Bengele HH, Mathias RS, Perkins JH, and Alexander EA. Urinary concentrating defect in the aged rat. Am J Physiol Renal Fluid Electrolyte Physiol 240: F147-F150, 1981.[Abstract/Free Full Text]
  7. Berger UV, Tsukaguchi H, and Hediger MA. Distribution of mRNA for the facilitated urea transporter UT3 in the rat nervous system. Anat Embryol (Berl) 197: 405-414, 1998.[Medline]
  8. Combet S, Geffroy N, Berthonaud V, Dick B, Teillet L, Verbavatz JM, Corman B, and Trinh-Trang-Tan MM. Correction of age-related polyuria by dDAVP: molecular analysis of aquaporins and urea transporters. Am J Physiol Renal Physiol 284: F199-F208, 2003.[Abstract/Free Full Text]
  9. Combet S, Teillet L, Geelen G, Pitrat B, Gobin R, Nielsen S, Trinh-Trang-Tan MM, Corman B, and Verbavatz JM. Food restriction prevents age-related polyuria by vasopressin-dependent recruitment of aquaporin-2. Am J Physiol Renal Physiol 281: F1123-F1131, 2001.[Abstract/Free Full Text]
  10. Corman B and Michel JB. Glomerular filtration, renal blood flow, and solute excretion in conscious aging rats. Am J Physiol Regul Integr Comp Physiol 253: R555-R560, 1987.[Abstract/Free Full Text]
  11. Cotrina ML and Nedergaard M. Astrocytes in the aging brain. J Neurosci Res 67: 1-10, 2002.[ISI][Medline]
  12. Donahue JL and Lowenthal DT. Nocturnal polyuria in the elderly person. Am J Med Sci 314: 232-238, 1997.[ISI][Medline]
  13. Ezzarani EA, Laulin JP, and Brudieux R. Effects of water deprivation on corticosterone production in the male Brattleboro rat genetically deprived of vasopressin. J Physiol (Paris) 80: 157-162, 1985.[Medline]
  14. Fenton RA, Cottingham CA, Stewart GS, Howorth A, Hewitt JA, and Smith CP. Structure and characterization of the mouse UT-A gene (Slc14a2). Am J Physiol Renal Physiol 282: F630-F638, 2002.[Abstract/Free Full Text]
  15. Flamion B, Spring KR, and Abramow M. Adaptation of inner medullary collecting duct to dehydration involves a paracellular pathway. Am J Physiol Renal Fluid Electrolyte Physiol 268: F53-F63, 1995.[Abstract/Free Full Text]
  16. Geelen G and Corman B. Relationship between vasopressin and renal concentrating ability in aging rats. Am J Physiol Regul Integr Comp Physiol 262: R826-R833, 1992.[Abstract/Free Full Text]
  17. Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 10: 230-237, 1999.[Abstract/Free Full Text]
  18. Kato A, Naruse M, Knepper MA, and Sands JM. Long-term regulation of inner medullary collecting duct urea transport in rat. J Am Soc Nephrol 9: 737-745, 1998.[Abstract]
  19. Keil LC and Severs WB. Reduction in plasma vasopressin levels of dehydrated rats following acute stress. Endocrinology 100: 30-38, 1977.[Abstract]
  20. Lim SW, Kim HY, Lee SB, Cha JH, Sands JM, Knepper MA, Madsen KM, and Kim J. Hydration status affects subcellular localization of UT-A and UT-B in rat kidney (Abstract). J Am Soc Nephrol 13: 68A, 2002.
  21. Macey RI and Yousef LW. Osmotic stability of red cells in renal circulation requires rapid urea transport. Am J Physiol Cell Physiol 254: C669-C674, 1988.[Abstract/Free Full Text]
  22. Manley GT, Fujimura M, Ma T, Noshita N, Filiz F, Bollen AW, Chan P, and Verkman AS. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med 6: 159-163, 2000.[ISI][Medline]
  23. Michimata M, Nogae S, Ohta M, Kaizuma S, Imai Y, Ito S, and Matsubara M. Topographic distribution of aquaporin 2 mRNA in the kidney of dehydrated rats. Exp Nephrol 8: 28-36, 2000.[ISI][Medline]
  24. Miller M. Influence of aging on vasopressin secretion and water regulation. In: Vasopressin, edited by Schrier RW. New York: Raven, 1985, p. 249-258.
  25. Miller M. Nocturnal polyuria in older people: pathophysiology and clinical implications. J Am Geriatr Soc 48: 1321-1329, 2000.[ISI][Medline]
  26. Mouton PR, Long JM, Lei DL, Howard V, Jucker M, Calhoun ME, and Ingram DK. Age and gender effects on microglia and astrocyte numbers in brains of mice. Brain Res 956: 30-35, 2002.[ISI][Medline]
  27. Murillo-Carretero MI, Ilundain AA, and Echevarria M. Regulation of aquaporin mRNA expression in rat kidney by water intake. J Am Soc Nephrol 10: 696-703, 1999.[Abstract/Free Full Text]
  28. Nakayama Y, Naruse M, Karakashian A, Peng T, Sands JM, and Bagnasco SM. Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter. Biochim Biophys Acta 1518: 19-26, 2001.[Medline]
  29. Naruse M, Klein JD, Ashkar ZM, Jacobs JD, and Sands JM. Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts. J Am Soc Nephrol 8: 517-523, 1997.[Abstract]
  30. Nielsen S, Nagelhus EA, Amiry-Moghaddam M, Bourque C, Agre P, and Ottersen OP. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 17: 171-180, 1997.[Abstract/Free Full Text]
  31. Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495-5500, 1996.[Abstract/Free Full Text]
  32. Olives B, Martial S, Mattei MG, Matassi G, Rousselet G, Ripoche P, Cartron JP, and Bailly P. Molecular characterization of a new urea transporter in the human kidney. FEBS Lett 386: 156-160, 1996.[ISI][Medline]
  33. Olives B, Mattei MG, Huet M, Neau P, Martial S, Cartron JP, and Bailly P. Kidd blood group and urea transport function of human erythrocytes are carried by the same protein. J Biol Chem 270: 15607-15610, 1995.[Abstract/Free Full Text]
  34. Olives B, Neau P, Bailly P, Hediger MA, Rousselet G, Cartron JP, and Ripoche P. Cloning and functional expression of a urea transporter from human bone marrow cells. J Biol Chem 269: 31649-31652, 1994.[Abstract/Free Full Text]
  35. Peng T, Sands JM, and Bagnasco SM. Glucocorticoids inhibit transcription and expression of the UT-A urea transporter gene. Am J Physiol Renal Physiol 282: F853-F858, 2002.[Abstract/Free Full Text]
  36. Popova NK, Naumenko KS, Maslova LN, Amstislavskaya TG, Melidi NN, Bulygina VV, and Ivanova LN. Hypothalamic tryptophan hydroxylase and the hypothalamic-pituitary-adrenocortical response to water deprivation and hydration in vasopressin-deficient and normal rats. Pflügers Arch 444: 372-377, 2002.[ISI][Medline]
  37. Preisser L, Teillet L, Aliotti S, Gobin R, Berthonaud V, Chevalier J, Corman B, and Verbavatz JM. Downregulation of aquaporin-2 and -3 in aging kidney is independent of V(2) vasopressin receptor. Am J Physiol Renal Physiol 279: F144-F152, 2000.[Abstract/Free Full Text]
  38. Sands JM. Molecular approaches to urea transporters. J Am Soc Nephrol 13: 2795-2806, 2002.[Abstract/Free Full Text]
  39. Sheehy CM, Perry PA, and Cromwell SL. Dehydration: biological considerations, age-related changes, and risk factors in older adults. Biol Res Nurs 1: 30-37, 1999.[Abstract/Free Full Text]
  40. Sladek CD, McNeill TH, Gregg CM, Blair ML, and Baggs RB. Vasopressin and renin response to dehydration in aged rats. Neurobiol Aging 2: 293-302, 1981.[ISI][Medline]
  41. Smith SJ. Neural signalling. Neuromodulatory astrocytes. Curr Biol 4: 807-810, 1994.[ISI][Medline]
  42. Steen B. Body water in the elderly—a review. J Nutr Health Aging 1: 142-145, 1997.[Medline]
  43. Swenson KL, Sands JM, Jacobs JD, and Sladek CD. Effect of aging on vasopressin and aquaporin responses to dehydration in Fischer 344-brown-Norway F1 rats. Am J Physiol Regul Integr Comp Physiol 273: R35-R40, 1997.[Abstract/Free Full Text]
  44. Terris JM, Knepper MA, and Wade JB. UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD. Am J Physiol Renal Physiol 280: F325-F332, 2001.[Abstract/Free Full Text]
  45. Timmer RT, Klein JD, Bagnasco SM, Doran JJ, Verlander JW, Gunn RB, and Sands JM. Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues. Am J Physiol Cell Physiol 281: C1318-C1325, 2001.[Abstract/Free Full Text]
  46. Trinh-Trang-Tan MM, Lasbennes F, Gane P, Roudier N, Ripoche P, Cartron J-P, and Bailly P. UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney. Am J Physiol Renal Physiol 283: F912-F922, 2002.[Abstract/Free Full Text]
  47. Trinh-Trang-Tan MM and Bankir L. Integrated function of urea transporters in the mammalian kidney. Exp Nephrol 6: 471-479, 1998.[ISI][Medline]
  48. Tsukaguchi H, Shayakul C, Berger UV, Tokui T, Brown D, and Hediger MA. Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis. J Clin Invest 99: 1506-1515, 1997.[ISI][Medline]
  49. Zhang C, Sands JM, and Klein JD. Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am J Physiol Renal Physiol 282: F85-F90, 2002.[Abstract/Free Full Text]



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