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【关键词】 vasopressin-induced
The Water and Salt Research Center, Institute of Anatomy, and Institute of Clinical Medicine, University of Aarhus, Aarhus, Denmark
Department of Biochemistry and Cell Biology, Kyungpook National University School of Medicine, Taegu, Korea
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Vasopressin and ANG II, which are known to play a major role in renal water and sodium reabsorption, are mainly coupled to the cAMP/PKA and phosphoinositide pathways, respectively. There is evidence for cross talk between these intracellular signaling pathways. We therefore hypothesized that vasopressin-induced water reabsorption could be attenuated by ANG II AT1 receptor blockade in rats. To address this, three protocols were used: 1) DDAVP treatment (20 ng/h sc for 7 days, n = 8); 2) DDAVP (20 ng/h sc for 7 days) and candesartan (1 mg?kg1?day1 sc for 7 days) cotreatment (n = 8); and 3) vehicle infusion as the control (n = 8). All rats were maintained on a NaCl-deficient diet (0.1 meq Na+?200 g body wt1?day1) during the experiment. DDAVP treatment alone resulted in a significant decrease in urine output (3.1 ± 0.2 ml/day) compared with controls (11.5 ± 2.2 ml/day, P < 0.05), whereas the urine output was significantly increased in response to DDAVP and candesartan cotreatment (9.8 ± 1.0 ml/day, P < 0.05). Consistent with this, rats cotreated with DDAVP and candesartan demonstrated decreased urine osmolality (1,319 ± 172 mosmol/kgH2O) compared with rats treated with DDAVP alone (3,476 ± 182 mosmol/kgH2O, P < 0.05). Semiquantitative immunoblotting revealed significantly decreased expression of medullary aquaporin-2 (AQP2) and AQP2 phosphorylated in the PKA phosphorylation consensus site Ser-256 (p-AQP2) in response to DDAVP and candesartan cotreatment compared with DDAVP treatment alone. In addition, cortical and medullary AQP1 was also downregulated. Fractional sodium excretion (FENa) and plasma potassium levels were markedly increased, and the expressions of the cortical type 3 Na+/H+ exchanger (NHE3), thiazide-sensitive Na-Cl cotransporter (NCC), and Na-K-ATPase were significantly decreased in response to DDAVP and candesartan cotreatment. Moreover, medullary type 1 bumetanide-sensitive Na-K-2Cl cotransporter expression showed a marked gel mobility shift from 160 to 180 kDa corresponding to enhanced glycosylation, whereas expression was unchanged. In conclusion, ANG II AT1 receptor blockade in DDAVP-treated rats was associated with decreased urine concentration and decreased AQP2 and AQP1 expression. Moreover, FENa was increased in parallel with decreased expression of NHE3, NCC, and Na-K-ATPase. These results suggest that ANG II AT1 receptor activation plays a significant role in regulating aquaporin and sodium transporter expression and modulating urine concentration in vivo.
aquaporin; calcium; cAMP; type 1 bumetanide-sensitive Na-K-2Cl cotransporter; urine concentration
VASOPRESSIN AND ANG II are importantly involved in the renal conservation of water and sodium (31, 32, 43, 45). Vasopressin is a peptide hormone that controls body fluid osmolality through the regulation of renal tubular water and sodium reabsorption (32). Its main sites of action in kidney tubules are the collecting duct and the thick ascending limb (TAL) of the loop of Henle, where vasopressin binds to the vasopressin V2 receptor and stimulates an increase in intracellular cAMP content via adenylyl cyclase (2, 31, 32, 56, 57). Subsequently, cAMP activates PKA, which phosphorylates various proteins including aquaporin-2 (AQP2) and the type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC-1) (10, 15, 16, 44).
In the collecting duct principal cells, AQP2 is then translocated from intracellular vesicles to the apical plasma membrane, thereby increasing osmotic water permeability (42). Moreover, transcriptional regulation of AQP2 in vivo is thought to be a result of a vasopressin-induced increase in intracellular cAMP levels (14, 21), which is capable of stimulating AQP2 gene transcription in cultured cells by acting through cAMP response element and AP-1 sites in the AQP2 promoter (22, 39). Binding of vasopressin to the V2 receptor is also associated with intracellular calcium mobilization in the inner medullary collecting duct cells (IMCDs), which triggers calmodulin-dependent regulatory processes within the cell (9). In the TAL of the loop of Henle, vasopressin V2 receptors are also present (2). Previous studies have demonstrated that vasopressin increases the intracellular level of cAMP in microdissected TAL segments (56, 57) as well as increases the rate of active NaCl absorption in isolated, perfused TALs (18, 20). Moreover, long-term elevation in circulating vasopressin levels in rats causes an increase in the medullary TAL (mTAL) NaCl transport rate (5) and expression of NKCC2 (28), thereby increasing urinary concentrating ability with the enhancement of countercurrent multiplication. Thus, as previously reviewed (31, 32, 43, 53), the factors that affect net water reabsorption in the kidney are 1) reabsorption of NaCl by the ascending limb (affected by both fluid delivery to and active transport capacity of the ascending limb); 2) water permeability of the medullary collecting duct; and 3) flow rate into the medullary collecting duct.
ANG II has known effects on the regulation of renal blood flow, glomerular filtration rate (GFR), and aldosterone secretion. Moreover, it is well established that ANG II increases sodium and bicarbonate reabsorption in the kidney proximal tubule via AT1 receptors. Recently, several studies have demonstrated that ANG II also has an important effect on the TAL and collecting duct in addition to the proximal tubules. In particular, 1) the TAL and collecting duct express ANG II receptor mRNA and protein (19, 54); 2) ANG II has an effect on the activity of Na-K(NH4+)-2Cl cotransport and bicarbonate transport in the isolated mTAL (1); 3) ANG II stimulates the activity of the epithelial sodium channel (ENaC) in the isolated cortical collecting duct (46) and induces vasopressin V2 receptor mRNA expression in cultured IMCDs (59); 4) ANG II regulates several signaling pathways including intracellular calcium, PKC activity, and metabolites of arachidonic acid in the TAL and collecting duct (7); and 5) we have recently demonstrated that ANG II infusion in rats increased the expression of the type 3 Na/H exchanger (NHE3) and NKCC2 in mTAL (35). Moreover, Beutler et al. (6) recently demonstrated that ANG II regulates ENaC expression, consistent with a role of ANG II in the regulation of collecting duct sodium transport (46). In addition, ANG II augments vasopressin-stimulated facilitated urea transport in the rat terminal IMCD (25), further suggesting a major role of ANG II in the regulation of urinary concentration.
The actions of vasopressin and ANG II require intracellular secondary messengers, which are mainly coupled to the cAMP/PKA and phosphoinositide pathways, respectively. Thus vasopressin induces an increase in intracellular cAMP levels (31, 32), whereas ANG II induces a rise in intracellular Ca2+ concentration ([Ca2+]) by inositol 1,4,5-triphosphate (7) and PKC activation by diacylglycerol (47). Interestingly, in vitro studies have demonstrated that ANG II potentiates vasopressin-dependent cAMP accumulation in Chinese hamster ovary cells transfected with cDNA of both AT1A and V2 receptors (30) and forskolin potentiates an ANG II-induced increase in intracellular [Ca2+] in cortical TAL (23). These results suggest cross talk between the signaling pathways of vasopressin and ANG II. Since intracellular trafficking of AQP2, expression of AQP2, and osmotic water permeability of the collecting duct principal cells are regulated by both intracellular cAMP production and intracellular [Ca2+] (9, 14, 52), we hypothesize that vasopressin and ANG II may have synergetic effects on urinary concentration in vivo. Thus it is possible that ANG II AT1 receptor blockade in rats treated with vasopressin and a NaCl-restricted diet (to induce a high plasma endogenous ANG II level) may reduce renal tubular water reabsorption and reduce the expression and/or targeting of vasopressin-regulated channels and transporters compared with rats treated with vasopressin and NaCl restriction without AT1 receptor blockade. The purposes of the present study are to examine 1) whether AT1 receptor blockade in rats cotreated with DDAVP (V2-receptor agonist) is associated with increased urine production and decreased urine concentration compared with rats treated with DDAVP alone; 2) whether AT1 receptor blockade in rats treated with DDAVP affects the expression of renal aquaporins [AQP1, AQP2, and p-AQP2 (AQP2 phosphorylated in the PKA phosphorylation consensus site Ser-256)]; and 3) whether AT1 receptor blockade in rats treated with DDAVP affects the expression of renal sodium transporters [NKCC2, NHE3, thiazide-sensitive Na-Cl cotransporter (NCC), and Na-K-ATPase].
METHODS
Experimental Protocols
DDAVP-treated rats vs. DDAVP- and candesartan-cotreated rats. Experiments were performed using male Munich-Wistar rats (200220 g, Taconic M&B, Ry, Denmark), which were maintained on a standard rodent diet (Altromin 1324, Lage, Germany). Control rats and experimental rats were chosen randomly and maintained in metabolic cages. Each rat received 15 g?200 g body wt1 (BW)?day1 of a low-sodium diet (Altromin C1036) during the entire experimental period. The estimated daily sodium intake in food was 0.1 meq Na+?200 g BW1?day1. They had free access to water intake. For DDAVP and/or candesartan infusion into normal Munich-Wistar rats, osmotic minipumps (model 2001, Alzet, Palo Alto, CA) were implanted subcutaneously in the neck of each rat. For implantation, osmotic minipumps were filled with DDAVP (V1005, Sigma) dissolved in physiological saline or candesartan (AT1 receptor blocker, a gift of Dr. Simon Clowes, Astra Pharmaceutical, Sodertalje, Sweden) dissolved in 0.02 M Na2CO3 in physiological saline. The pumps were equilibrated with physiological saline at 37°C for 4 h before insertion. The dose of candesartan (when given in drinking water) has been shown to be sufficient to block the rise in blood pressure resulting from long-term infusion of ANG II (24). The animal protocols have been approved by the boards of the Institute of Anatomy and Institute of Clinical Medicine, University of Aarhus, according to the licenses for use of experimental animals issued by the Danish Ministry of Justice.
Group 1 (DDAVP-treated group: V group, n = 8) rats were given DDAVP at a dose of 20 ng/h for 7 days (sc) (28) and a low-sodium diet (0.1 meq Na+?200 g BW1?day1).
Group 2 (DDAVP- and candesartan-cotreated group: VC group, n = 8) rats were given both DDAVP at a dose of 20 ng/h for 7 days (sc) (28) and candesartan at a dose of 1 mg?kg1?day1 for 7 days (sc) (6) and a low-sodium diet (0.1 meq Na+?200 g BW1?day1).
Group 3 (vehicle-treated group: controls, n = 8) rats were given saline/Na2CO3 vehicle and a low-sodium diet (0.1 meq Na+?200 g BW1?day1).
Clearance Studies
The rats were maintained in metabolic cages, and daily 24-h urine output and water intake were measured during the experimental period. After 7 days of DDAVP and/or candesartan treatment, all rats were anesthetized under halothane inhalation and blood was collected from the inferior vena cava at the time of death. The plasma and urinary concentrations of creatinine and urea nitrogen and the plasma concentrations of sodium and potassium were determined (Vitros 950, Johnson & Johnson). The concentrations of urinary sodium and potassium were determined by standard flame photometry (Eppendorf FCM6341). The osmolality of plasma and urine was determined by freezing-point depression (Osmomat 030-D, Gonotec, Berlin, Germany).
Measurement of Plasma Aldosterone
Blood samples were drawn in EDTA glass vials. Immunoreactive aldosterone was measured by a radioimmunoassay method (Diagnostic System Laboratories, Webster, TX). Using a rabbit-anti-aldosterone-antibody, a 125I-aldosterone radioimmunoassay was performed by incubation of plasma samples in precoated tubes. The lowest detectable level was 50 pmol/l.
Membrane Fractionation and Immunoblotting
All rats were killed under light halothane anesthesia, and the right kidneys were rapidly removed. Each kidney was dissected into the cortex/outer stripe of outer medulla (cortex+OSOM), inner stripe of outer medulla (ISOM), and inner medulla. The dissected renal cortex+OSOM, ISOM, and inner medulla were homogenized (Ultra-Turrax T8 homogenizer, IKA Labortechnik, Staufen, Germany) in ice-cold isolation solution containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2. To remove whole cells, nuclei and mitochondria, the homogenates were centrifuged at 4,000 g for 15 min at 4°C and the supernatant was pipetted off and kept on ice for further processing. The total protein concentration was measured (Pierce BCA protein assay reagent kit, Pierce, Rockford, IL), and all samples were adjusted with isolation solution to reach the same final protein concentrations, solubilized at 65°C for 15 min in Laemmli sample buffer, and then stored at 20°C. To confirm equal loading of protein, an initial gel was stained with Coomassie blue dye as described previously (41). SDS-PAGE was performed on 9 or 12% polyacrylamide gels. The proteins were transferred from the gel electrophoretically (Bio-Rad Mini Protean II) to nitrocellulose membranes (Hybond ECL RPN3032D, Amersham Pharmacia Biotech, Little Chalfont, UK). After transfer, the blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with primary antibodies. Immunoblotting was performed using anti-rat AQP1 (55), anti-rat AQP2 (42), anti-rat p-AQP2 (10), anti-rat NHE3 (27, 35, 41), anti-rat NKCC2 (12, 27, 28, 35, 41), anti-1-subunit of Na-K-ATPase (35, 41), or anti-rat NCC antibodies (35, 41). The sites of an antibody-antigen reaction were visualized with horseradish peroxidase-conjugated secondary antibodies (P447 or P448, diluted 1:3,000; DAKO, Glostrup, Denmark) with an enhanced chemiluminescence (ECL) system and exposure to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech). The band densities were quantitated by scanning the films, and the density was calculated as a fraction of the mean control value for that gel.
Deglycosylation
From each rat, 18 μl of ISOM protein (supernatant from 4,000-g centrifugation for 15 min at 4°C) and 2 μl of 10% SDS (wt/vol in dissection buffer) were mixed and heated to 95°C for 5 min. After cooling to room temperature, 180 μl of 1% Triton X-100 (vol/vol in dissection buffer, T9284, Sigma) were added and mixed thoroughly before the sample was aliquoted into two aliquots of 100 μl each. For paired aliquots, 10 μl (1 unit/μl) of N-glycosidase F (PNGase F, recombinant, catalogue no. 1 365 177, Roche Diagnostics, Nonnenwald, Germany) were added to one, whereas 10 μl of dissection buffer were added to the other as a control. Samples were left overnight at room temperature, and the reaction was stopped by solubilizing protein in Laemmli sample buffer and heating to 65°C for 15 min (36).
Immunohistochemistry
Left kidneys were fixed by retrograde perfusion via the aorta with 3% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. Immunolabeling was performed on sections from the paraffin-embedded preparation (2-μm thickness) using methods described previously in detail (41).
Statistical Analyses
Values are presented as means ± SE. Data were analyzed by one-way ANOVA followed by Bonferroni's multiple-comparisons test. Multiple-comparisons tests were only applied when a significant difference was determined in the ANOVA (P < 0.05).
RESULTS
Rats Cotreated with Both DDAVP and Candesartan Have Increased Urine Output and Decreased Urine Osmolality Compared with Rats Treated with DDAVP Alone
Three study groups (n = 8, respectively) were used, and all rats in the three groups were placed on a low-sodium-containing diet. One-way ANOVA with a multiple-comparisons test revealed that rats treated with DDAVP (V group) exhibited decreased urine output and increased urine osmolality (Table 1, Fig. 1). In contrast, during the entire experimental period, the urine output in rats cotreated with DDAVP and candesartan (VC group) was significantly higher than that of rats receiving DDAVP alone (V group, Fig. 1). On the last day of the experiment, AT1 receptor blockade in rats treated with DDAVP (VC group) resulted in an increase in urine output (9.8 ± 1.0 vs. 3.1 ± 0.2 ml/day at day 7, P < 0.05; Table 1, Fig. 1) and decreased urine osmolality (1,319 ± 172 vs. 3,476 ± 182 mosmol/kgH2O at day 7, P < 0.05; Table 1) compared with rats in the V group. Compared with the vehicle-infused control rats, the urine output in the VC group returned to the similar levels of the controls after day 3 (Fig. 1). Thus candesartan cotreatment did not prevent the acute decrease in urine production in response to DDAVP but resulted in a gradual increase in urine production over 7 days.
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On the last day of the experiment, the urine-to-plasma osmolality ratio (3.6 ± 0.5 vs. 11.9 ± 0.7, P < 0.05; Table 1) and solute-free water reabsorption (22.7 ± 1.3 vs. 33.0 ± 1.0 ml/day, P < 0.05; Table 1) were also significantly lower in the VC group compared with the V group, compatible with the observed high urine output. In contrast, these functional parameters in the VC group were similar to the controls, suggesting that angiotensin II AT1 receptor blockade significantly decreased the DDAVP-induced urine concentration.
Renal Sodium Handling, Creatinine Clearance, Urea Nitrogen Excretion, and Plasma Sodium, Potassium, and Urea Nitrogen Levels Are Altered in Rats Cotreated with DDAVP and Candesartan
Fractional urinary excretion of sodium (FENa) and plasma potassium levels were significantly increased in rats cotreated with DDAVP and candesartan (VC group, Table 1). In contrast, urinary sodium excretion rates were similar between the two groups (VC group vs. V group), consistent with equal dietary sodium intake and steady state and possibly due to a decreased GFR in the VC group. GFR, measured by creatinine clearance, was decreased compared with the V group (0.75 ± 0.08 vs. 1.35 ± 0.06 ml/min, P < 0.05; Table 1) or the controls (0.75 ± 0.08 vs. 1.39 ± 0.08 ml/min, P < 0.05; Table 1), consistent with previous studies (8). Plasma urea nitrogen levels in both the V group and VC group were significantly increased compared with the controls (Table 1), and the increase was more prominent in the VC group, in which high plasma osmolality was observed (Table 1). Consistent with this, fractional excretion of urea nitrogen was decreased in both the V group and the VC group, and the more prominent decrease was seen in the VC group (Table 1).
Candesartan Cotreatment Reduces Expression of AQP2 and p-AQP2 in Kidney Inner Medulla and ISOM
Semiquantitative immunoblotting of proteins prepared from the kidney inner medulla revealed that AQP2 expression was significantly increased in response to vasopressin V2-receptor agonist DDAVP treatment for 7 days (144 ± 12% in the V group vs. 100 ± 8% in controls, P < 0.05; Fig. 2, A and C, Table 2), whereas AQP2 expression was unchanged in response to DDAVP and candesartan cotreatment for 7 days [110 ± 2% in the VC group vs. 100 ± 8% in controls, not significant (NS); Fig. 2, A and C, Table 2] compared with controls. Moreover, inner medullary p-AQP2 expression was significantly increased in response to 7 days of DDAVP treatment (175 ± 21% in the V group vs. 100 ± 15% in controls, P < 0.05; Fig. 2, B and D, Table 2), whereas p-AQP2 expression was unchanged in response to 7 days of DDAVP and candesartan cotreatment (105 ± 13% in the VC group vs. 100 ± 15% in controls, NS; Fig. 2, B and D, Table 2) compared with controls. Thus AT1 receptor blockade in rats treated with DDAVP appeared to prevent the DDAVP-induced upregulation of AQP2 and p-AQP2 in the inner medulla and thereby antagonized the effect of DDAVP.
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Immunoperoxidase labeling of p-AQP2 in the kidney inner medulla was associated with the apical domains of IMCDs (arrows in Fig. 3, AC), as previously demonstrated (10). Consistent with the semiquantitative immunoblotting of p-AQP2 expression (Fig. 2, B and D), strong apical immunoperoxidase labeling of p-AQP2 was observed in the V group (arrows in Fig. 3A), whereas candesartan cotreatment reduced the labeling of p-AQP2 (arrows in Fig. 3B) and the labeling was similar to the labeling seen in the controls (arrows in Fig. 3C).
In the ISOM, AQP2 expression was significantly increased in both the V group (400 ± 23 vs. 100 ± 6% in controls, P < 0.05; Fig. 4, B and D, Table 3) and the VC group (289 ± 23 vs. 100 ± 6% in controls, P < 0.05; Fig. 4, B and D, Table 3) compared with controls, respectively. The upregulation of AQP2 seen in the VC group, however, was less prominent compared with the increased AQP2 expression in the V group (Fig. 4, B and D, P < 0.05). p-AQP2 expression in the ISOM was also significantly increased in both the V group (227 ± 12 vs. 100 ± 11% in controls, P < 0.05; Table 3) and the VC group (226 ± 20 vs. 100 ± 11% in controls, P < 0.05; Table 3), and no difference in p-AQP2 expression was observed between the V group and the VC group.
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In contrast to the changes in AQP2 expression seen in the inner medulla and ISOM between the V group and the VC group, there were no changes in AQP2 and p-AQP2 expression in proteins prepared from the cortex + OSOM combined. Semiquantitative immunoblotting of proteins prepared from cortex + OSOM combined revealed that AQP2 expression was similarly high in both the V group (202 ± 4 vs. 100 ± 6% in controls, P < 0.05; Fig. 4, A and C, Table 4) and the VC group (223 ± 11 vs. 100 ± 6% in controls, P < 0.05; Fig. 4, A and C, Table 4) compared with controls, respectively. Moreover, the expression of p-AQP2 in the cortex and OSOM was also increased to the same extent in both the V group (216 ± 10 vs. 100 ± 6% in controls, P < 0.05; Table 4) and VC group (245 ± 18 vs. 100 ± 6% in controls, P < 0.05; Table 4) compared with controls, respectively.
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Candesartan Cotreatment Decreases Expression of AQP1 in Kidney Cortex and Outer Medulla
Semiquantitative immunoblotting of proteins prepared from the kidney cortex and the OSOM revealed that AQP1 expression was significantly decreased in the VC group (63 ± 6 vs. 100 ± 3% in controls, P < 0.05; Fig. 5, A and C, Table 4), whereas AQP1 expression was unchanged in the V group (110 ± 6 vs. 100 ± 3% in controls, NS; Fig. 5, A and C, Table 4) compared with controls, respectively. Moreover, in the ISOM, the expression of AQP1 was also significantly decreased in the VC group (40 ± 5 vs. 100 ± 7% in controls, P < 0.05; Fig. 5, B and D, Table 3), whereas AQP1 expression was unchanged in the V group (82 ± 10 vs. 100 ± 7% in controls, NS; Fig. 5, B and D, Table 3). In the inner medulla, AQP1 expression was unchanged in both groups compared with controls (Table 2).
Candesartan Cotreatment Does Not Change NKCC2 Expression But Changes Molecular Mass
Semiquantitative immunoblotting revealed that NKCC2 expression in the ISOM was unchanged in response to DDAVP treatment (104 ± 13% in the V group vs. 100 ± 17% in controls, NS; Fig. 6, B and D, Table 3) or to DDAVP and candesartan cotreatment (122 ± 12% in the VC group vs. 100 ± 17% in controls, NS; Fig. 6, B and D, Table 3) compared with controls, respectively. Consistent with this, immunoperoxidase microscopy demonstrated that the apical expression and subcellular localization of NKCC2 in mTAL were not different (not shown). However, immunoblotting revealed that candesartan cotreatment was associated with a marked gel mobility shift from 160 to 180 kDa compared with either the V group or controls (Fig. 6B). Moreover, in the cortex + OSOM, NKCC2 expression was unchanged in both the V group (102 ± 7 vs. 100 ± 6% in controls, NS; Fig. 6, A and C, Table 4) and VC group (92 ± 17 vs. 100 ± 6% in controls, NS; Fig. 6, A and C, Table 4). Similar to the changes in the ISOM, NKCC2 expression in the VC group was associated with a marked gel mobility shift in the cortex and OSOM compared with either the V group or controls (Fig. 6A).
To examine whether the shift in mobility was due to alterations in the glycosylation of NKCC2, proteins from the ISOM were treated with PNGase F for 24 h at room temperature. The results revealed that PNGase F treatment was associated with a reduction in the molecular mass to 120 kDa (Fig. 7), corresponding to the expected size of NKCC2 (37). Thus candesartan cotreatment induces a differential glycosylation compared with that seen without the cotreatment. Whether these forms have distinct functional characteristics and/or represent different stages of biosynthesis or degradation awaits future investigation.
Decreased Expression of NHE3 in Kidney Cortex and OSOM in Response to Candesartan Cotreatment
Semiquantitative immunoblotting of proteins prepared from the kidney cortex + OSOM revealed that candesartan cotreatment (VC group) was associated with a significant decrease in NHE3 expression (56 ± 4% in the VC group vs. 100 ± 4% in controls, P < 0.05; Fig. 8, A and C, Table 4). In contrast, the expression of NHE3 in kidney cortex and OSOM in the V group, which only received DDAVP, did not change significantly (113 ± 13% in the V group vs. 100 ± 4% in controls, NS; Fig. 8, A and C, Table 4). This finding was further demonstrated by immunohistochemistry. Immunoperoxidase microscopy in kidney cortex and OSOM of control rats revealed NHE3 immunolabeling of the apical plasma membrane domains of S1 and S2 segments of convoluted proximal tubules (arrows in Fig. 9, AC) with weaker staining of the S3 straight proximal tubule segments (not shown), as previously demonstrated (35). In addition, NHE3 labeling was associated with the apical plasma membrane domains of the cortical TAL (Fig. 9, A and B, indicated as C) in the kidney cortex. Immunoperoxidase microscopy demonstrated that candesartan cotreatment (VC group) resulted in a decrease in NHE3 immunolabeling of the apical plasma membrane domains of the proximal tubule (arrows in Fig. 9B) compared with either the V group (arrows in Fig. 9A) or controls (arrows in Fig. 9C). In contrast, the labeling of NHE3 in the apical plasma membrane domains of the cortical TAL was unchanged in response to candesartan cotreatment (indicated as C in Fig. 9B) compared with either the V group (indicated as C in Fig. 9A) or controls (not shown), suggesting that the decreased NHE3 expression in the cortex + OSOM was mainly due to the decreased expression in the proximal tubule. On the contrary, immunoblotting of proteins prepared from the ISOM (Fig. 8) and immunocytochemistry (not shown) revealed that NHE3 expression in the ISOM was unchanged among groups (Fig. 8, B and D, Table 3).
Candesartan Cotreatment Results in Decreased Expression of NCC and Na-K-ATPase in Kidney Cortex and OSOM
In the kidney cortex + OSOM, semiquantitative immunoblotting demonstrated that candesartan cotreatment was associated with significantly decreased NCC expression (58 ± 4% in the VC group vs. 100 ± 1% in controls, P < 0.05; Fig. 10, A and C, Table 4) and Na-K-ATPase expression (37 ± 6% in the VC group vs. 100 ± 5% in controls, P < 0.05; Fig. 10, B and D, Table 4) compared with controls. In contrast, DDAVP treatment alone (V group) was associated with unchanged expression of NCC and Na-K-ATPase in the cortex and OSOM (Fig. 10, AD, Table 4).
Immunoperoxidase microscopy of the kidney cortex from control rats (Fig. 9F) demonstrated that the proximal tubules exhibited Na-K-ATPase labeling of basolateral plasma membranes ( in Fig. 9F) and cortical TAL and distal convoluted tubules showed very intense basolateral labeling (Fig. 9F). Immunoperoxidase microscopy demonstrated decreased Na-K-ATPase immunolabeling of the basolateral plasma membranes of the proximal tubules in the VC group ( in Fig. 9E) compared with either the V group ( in Fig. 9D) or controls ( in Fig. 9F), consistent with the immunoblotting data (Fig. 10, B and D). In contrast, the basolateral labeling of Na-K-ATPase in the cortical TAL and distal convoluted tubules appeared unchanged in response to candesartan cotreatment (Fig. 9E, VC group) compared with either the V group (Fig. 9D) or controls (Fig. 9F). Thus the reduction in immunolabeling of Na-K-ATPase was primarily due to reduction in the proximal tubule. Moreover, Na-K-ATPase expression was significantly reduced in the ISOM in response to candesartan cotreatment compared with the group that received DDAVP alone (77 ± 14% of control levels vs. 114 ± 7% of control levels, P < 0.05; Table 3).
DISCUSSION
In the present study, we demonstrated that pharmacological blockade of the AT1 receptor in rats cotreated with DDAVP and dietary NaCl restriction (to induce high plasma endogenous ANG II levels) were associated with significant changes in urinary water and sodium excretion in parallel with changes in the expression of renal aquaporins and sodium transporters. In brief, AT1 receptor blockade with candesartan 1) induced a time-dependent progressive increase in urine production and a decrease in urine osmolality; 2) prevented upregulation of inner medullary AQP2 and p-AQP2 expression in response to long-term DDAVP administration (thus candesartan treatment effectively reduced the expression); 3) induced a decrease in the expression of AQP1; 4) induced no changes in the expression of medullary NKCC2 but resulted in a marked change in molecular mass due to changes in glycosylation; and 5) induced a significant reduction in the expression of NHE3, NCC, and Na-K-ATPase in the kidney cortex. The results suggest that ANG II-stimulated AT1 receptor activation may play a role in the regulation of collecting duct vasopressin-regulated AQP2 expression (especially in the inner medulla) and urine concentration in vivo, in addition to regulating the expression of major renal sodium transporters and urea transporters as previously demonstrated (6, 25, 35).
Occupation of ANG II AT1 Receptor Plays a Role in Regulation of Vasopressin-Regulated AQP2 Expression in Collecting Ducts and Urine Concentration In Vivo
Urinary concentration and dilution depend on the presence of a discrete segmental distribution of transport properties along the renal tubule, and urinary concentration depends on 1) a hypertonic medullary interstitium, which is generated by active NaCl reabsorption as a consequence of countercurrent multiplication in water-impermeable nephron segments; 2) high water permeability (constitutive or vasopressin regulated) in other renal tubular segments for osmotic equilibration, which chiefly depends on the expression of aquaporins; and 3) the flow rate of tubule fluid entering the medullary collecting ducts. Thus defects in any of these mechanisms would be predicted to be associated with urinary concentrating defects.
The collecting duct represents the final site for the control of water excretion into the urine. Water permeability of the collecting duct is tightly regulated, under the control of vasopressin, which causes a dramatic increase in collecting duct water permeability, allowing reabsorption of water from the tubular fluid down an osmotic gradient. We here confirmed that long-term DDAVP treatment upregulates inner medullary AQP2 and p-AQP2 expression, consistent with previous studies (10, 48). In contrast, cotreatment with the AT1-receptor antagonist candesartan prevented the increase in the expression of inner medullary AQP2 and p-AQP2 in response to the long-term DDAVP treatment. On the contrary, expression of outer medullary NKCC2, NHE3, and Na-K-ATPase, which are chiefly involved in the active reabsorption of NaCl by the mTAL to maintain the countercurrent multiplication, was unchanged in response to DDAVP treatment, in both the presence and the absence of candesartan. This suggests that the progressive blunting of urine concentration in response to DDAVP seen in the rats cotreated with the AT1 receptor blocker candesartan was primarily due to the prevention of a DDAVP-mediated increase in the osmotic water permeability in the outer medullary collecting ducts and IMCDs despite long-term DDAVP treatment. This is evidenced by the maintained (not increased) expression of AQP2 and p-AQP2, respectively.
The effects of vasopressin and ANG II are mainly coupled to the cAMP/PKA and phosphoinositide pathways, respectively. Thus vasopressin induces an increase in intracellular cAMP levels, whereas ANG II induces a rise in intracellular [Ca2+]. Recently, Chou et al. (9) demonstrated that vasopressin, acting through vasopressin V2 receptor, also causes intracellular [Ca2+] mobilization in the isolated IMCD as a result of release of [Ca2+] via the type 1 ryanodine receptor. Moreover, it has been shown that ANG II (107 mol/l) also induced an increase in intracellular [Ca2+] in microdissected IMCD, although the increase in intracellular [Ca2+] was larger in the proximal tubule and the thick ascending limb (7). Since regulation of intracellular AQP2 trafficking, AQP2 protein expression, and osmotic water permeability in the collecting duct principal cells involves intracellular cAMP production and [Ca2+] levels (9, 14, 52), we hypothesize that ANG II AT1 receptor blockade could reduce the synergetic effects of ANG II on the vasopressin-induced increase in intracellular cAMP and [Ca2+] levels. This hypothesis was further supported by previous in vitro studies demonstrating cross talk between the signaling pathways of vasopressin and ANG II (23, 30). Consistent with this, we found that inner medullary expression of AQP2 and p-AQP2 and urine osmolality were not increased in response to long-term DDAVP administration in NaCl-restricted rats in the presence of AT1 receptor blocker candesartan cotreatment. This indicates that the occupation of the ANG II AT1 receptor plays a role in the regulation of vasopressin-regulated AQP2 expression in collecting ducts and urine concentration in vivo. Moreover, the dual ANG II/AVP receptor, which is a novel receptor coupled to adenylate cyclase that responds with equal sensitivity to ANG II and AVP, has been isolated (49). The renal immunocytochemical distribution of the ANG II/AVP receptor to IMCDs also strongly suggests the role of ANG II in the regulation of vasopressin-regulated AQP2 expression in the collecting ducts and urine concentration in vivo (17).
A question arises as to whether the observed absence of an increase in inner medullary AQP2 and p-AQP2 expression in response to DDAVP treatment in rats cotreated with candesartan (VC group) may reflect the effect of an increased tubular flow rate in the IMCD. An increased flow rate in the collecting duct could potentially be caused in part by the decreased expression of AQP1 in the proximal nephron, NHE3 and Na-K-ATPase in the proximal tubule, and NCC in the distal convoluted tubule. Previously, we demonstrated that rats with diabetes mellitus, which are known to have distinct glomerular hyperfiltration, osmotic diuresis and polyuria, and a significant elevation of plasma vasopressin levels, exhibited a sixfold increase in urine output as well as a twofold increase in inner medullary AQP2 expression as a compensatory process (40). Thus this finding suggests that the changes in tubular flow rate in the IMCD per se do not decrease AQP2 expression. Moreover, rats treated with furosemide for 5 days showed a sevenfold increase in urine output but no changes in inner medullary AQP2 expression (38). This also suggests that the changes in medullary osmolality and tubular flow are not the major factors causing changes in AQP2 expression.
Decreased Expression of Renal AQP1 in Kidney Cortex and Outer Medulla in Response to DDAVP in the Presence of AT1 Receptor Blockade
We demonstrated that AQP1 expression in both the cortex and outer medulla was significantly decreased in response to DDAVP and candesartan cotreatment compared with either DDAVP-treated rats or control rats, all of which were on a NaCl-deficient diet. AQP1 is highly abundant in the proximal tubule and descending thin limb, and previous studies have emphasized its critical role in the constitutive water reabsorption in these segments (43). In particular, AQP1-transfected LLC-PK1 cells exhibited constitutively high water permeability that was not regulated by vasopressin (26). Consistent with this, we have previously demonstrated that renal AQP1 expression was not altered in rats with the severe nephrogenic diabetes insipidus induced by chronic lithium treatment (34), in which plasma vasopressin and aldosterone levels are significantly elevated and AQP2 and AQP3 expression was significantly decreased (34, 41). This suggests that the changes in the expression of AQP1 could be a direct consequence of AT1 receptor blockade. Consistent with this, the expression levels of two additional proximal tubule sodium transporters, i.e., NHE3 and Na-K-ATPase, are also downregulated in response to candesartan cotreatment in DDAVP-infused rats. Moreover, the downregulation of NHE3 and Na-K-ATPase was also observed in our parallel study examining the effect of AT1 receptor blocker candesartan treatment on the expression of renal sodium transporters in rats that received a very low-sodium diet without DDAVP infusion (Turban S, unpublished observations), where GFR was significantly decreased.
On the other hand, it may be speculated that the decreased expression of AQP1, NHE3, and Na-K-ATPase in the proximal tubule could be caused by the decreased GFR as observed in response to candesartan treatment. The proximal tubule is the site of reabsorption of approximately two-thirds of the NaCl that enters the tubular fluid by glomerular filtration and therefore is the main energy-expending compartment of the kidney. Thus structural and functional adaptation of the proximal tubule may potentially occur in response to the changes in single-nephron GFR and tubular fluid flow rate. For instance, this is known to occur in remnant kidneys induced by nephrectomy (33). Consistent with this view, our parallel study (Turban S, unpublished observations) demonstrated that a 2-day infusion of candesartan in rats treated with a relatively low-sodium diet (0.5 meq Na+?200 g BW1?day1) demonstrated unchanged GFR and unchanged cortical expression of NHE3 (but with decreased expression of NHE3 in the brush-border membrane preparation). In contrast, a 2-day infusion of candesartan in rats treated with a very low-sodium diet (0.03 meq Na+?200 g BW1?day1) was associated with decreased GFR and decreased cortical expression of NHE3. However, the underlying mechanisms for the decreased expression of AQP1 in the proximal tubules and in the descending thin limbs as well as proximal tubule NHE3 in response to AT1 receptor blockade are still unclear, and future studies will be needed to establish them.
Unchanged NKCC2 Expression But Marked Gel Mobility Shift in VC Group
It has been demonstrated that protein expression of NKCC2 in the TAL is regulated and this appears to play a significant role in the urinary concentration mechanism. An increase in the delivery of NaCl to the loop of Henle by chronic oral saline loading (12) or DDAVP treatment in Brattleboro rats (28) has been demonstrated to upregulate NKCC2 levels, whereas hypokalemia-induced nephrogenic diabetes insipidus (13) or parathyroid hormone-induced hypercalcemia (58) was associated with reduced NKCC2 expression along with decreased urine concentration. Vasopressin is generally believed to play an important role in NaCl reabsorption in the TAL, which could be mediated by V2 receptors, adenylyl cylase, and cAMP (5, 16, 31). Nevertheless, in the present study we demonstrated that NKCC2 expression in both the outer medulla and cortex was unchanged in response to DDAVP treatment in NaCl-restricted rats in both the absence and presence of candesartan cotreatment compared with controls, respectively. The increased expression of outer medullary AQP2 in response to DDAVP in both groups provided a positive control for the effect of DDAVP. The observation that DDAVP treatment did not alter NKCC2 expression is compatible with a previous study (12) showing unchanged NKCC2 protein expression in the outer medulla and cortex in response to 5 days of AVP administration to Brattleboro rats. Thus it may be possible that chronic regulation of NaCl transport by vasopressin mainly involves mechanisms other than regulation of NKCC2 expression, although the results cannot completely rule out the opposite effect of other intervening regulatory factors (e.g., altered sympathetic nerve activity) on the upregulation of NKCC2 expression possibly induced by vasopressin. Moreover, the observed lack of effect of DDAVP on NKCC2 expression could be attributed to the difference in rat strains, which may involve different adenylate cyclase responses to vasopressin (29).
Interestingly, NKCC2 expression in the VC group was associated with a marked gel mobility shift from 160 to 180 kDa compared with either the V group or controls in both the outer medulla and cortex. The increase in molecular mass is likely to be a consequence of an alteration in glycosylation of the NKCC2 protein, because the bands with different molecular mass seen in the V group, VC group, and controls actually migrated on SDS gels to bands with same size of 120 kDa after PNGase F deglycosylation. This finding was consistent with a previous study demonstrating that vasopressin-treated Sprague-Dawley rats receiving furosemide (12) were associated with an increase in the average molecular mass of NKCC2 by 9 kDa in both the outer medulla and cortex. One possible explanation for an alteration in glycosylation has been proposed by Barasch and Al-Awqati (4), pointing out that some glycosylating enzymes, most notably sialyl transferase, are pH sensitive. Thus it is possible that an alteration in glycosylation of NKCC2 seen in the candesartan-treated rats (VC group) may be due to the changes in intracellular pH of the TAL cells. However, it is unknown whether these differentially glycosylated forms have different functional characteristics and/or represented different stages of biosynthesis or degradation.
Decreased Cortical Expression of NHE3 in VC Group
We have previously demonstrated that NHE3 immunolabeling in the brush border of the proximal tubules was markedly enhanced in ANG II-treated rats, although the overall protein expression of NHE3 and Na-K-ATPase remained unchanged in the kidney cortex (35). In the present study, ANG II AT1 receptor blockade reduced immunolabeling of NHE3 in the proximal tubule brush border as well as NHE3 expression in the kidney cortex. Consistent with the proposed roles of the NHE3 in kidney tubules, the proximal convoluted tubules from NHE3 gene knockout mice had a marked decrease in fluid and bicarbonate absorption (51). These findings therefore indicate that the decreased NHE3 expression in the cortex and OSOM in the VC group may partly contribute to increased urine output and FENa compared with either the V group or controls.
Potential Role of ANG II in Urea Equlibration and Urinary Concentration
Arginine vasopressin stimulates urea transport in perfused rat terminal IMCDs (50). On the contrary, ANG II has no effect on basal facilitated urea permeability but increases vasopressin-stimulated urea permeability in rat terminal IMCDs and 32P incorporation into both 117- and 97-kDa UT-A1 proteins via a PKC-mediated effect (25). This indicates a physiological role of ANG II in the urinary concentrating mechanism by increasing urea permeability in response to vasopressin in the IMCDs. Moreover, this is consistent with our data showing the AT1 receptor blockade in rats treated with DDAVP reduces renal tubular water reabsorption and urinary concentration compared with rats treated with vasopressin alone without AT1 receptor blockade. However, the observed markedly increased plasma urea nitrogen levels and decreased fractional excretion of urea nitrogen in rats cotreated with DDAVP and candesartan despite the lack of differences in body weight and food intake need to be evaluated in a future study, in parallel with investigating the changes in renal urea transporter expression.
Summary and Perspective
In the present study, we demonstrated that pharmacological blockade of the AT1 receptor in long-term DDAVP-treated rats was associated with reduced urine concentration and prevention of upregulation of AQP2 and p-AQP2 expression in the inner medulla. In addition, cortical and medullary AQP1 expression was significantly decreased. Medullary NKCC2 expression was unchanged but showed a marked gel mobility shift from 160 to 180 kDa. The increase in molecular mass observed in the VC group was likely to be a consequence of an alteration in glycosylation of the NKCC2 protein. Finally, cortical expression of NHE3, NCC, and Na-K-ATPase was decreased in response to AT1 receptor blockade. The results suggest that the occupation of the ANG II AT1 receptor may play a role in regulating collecting duct vasopressin-regulated AQP2 expression (especially in the inner medulla) and urine concentration in vivo, in addition to regulating the expression of major renal sodium transporters in the proximal tubule and distal tubule, including collecting ducts.
The actions of ANG II are mediated by AT1 as well as AT2 receptors. In the present study, we did not examine the potential role of the AT2 receptor in urinary concentration, which may be stimulated by the increase in plasma ANG II levels through AT1 receptor blockade (11). Because AT2-mediated counterregulatory vasodilatation can oppose AT1-mediated vasoconstriction, the stimulated AT2-mediated action could contribute to altered renal perfusion and renal cortical or medullary circulation (3). The effect of AT2-mediated action on urinary concentration needs to be examined.
GRANTS
The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Support for this study was provided by the Karen Elise Jensen Foundation, Human Frontier Science Program, Novo Nordic Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, University of Aarhus, European Commission (QRLT 2000 00778 and QRLT 2000 00987), the Advanced Medical Technology Cluster for Diagnosis and Prediction at Kyungpook National University from MOCIE (T.-H. Kwon), and the intramural budget of the National Heart, Lung, and Blood Institute, National Institutes of Health.
ACKNOWLEDGMENTS
The authors thank Helle Hyer, Lotte Vallentin Holbech, Ida Maria Jalk, Inger Merete Paulsen, Zhila Nikrozi, Mette Vistisen, Gitte Kall, and Dorte Wulff for expert technical assistance.
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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