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作者：Lone Brønd, Niels Hadrup, Nanna Salling, Malene Torp, Martin Græbe, Sten Christensen, Søren Nielsen, and Thomas E. N. Jonassen作者单位：1 Department of Pharmacology, University of Copenhagen, DK-2200 Copenhagen N; and 2 The Water and Salt Research Center, Institute of Anatomy (Building 233), University of Aarhus, DK-8000 Aarhus C, Denmark
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【摘要】
) g+ c7 r5 B9 Z4 T* M Vasopressin (AVP) stimulates collecting duct water reabsorption through cAMP-mediated membrane targeting and increased expression of the aquaporin-2 (AQP2) water channel. Rats with liver cirrhosis induced by common bile duct ligation (CBL) show decreased protein expression of AQP2 despite increased plasma concentrations of AVP. The present study was conducted to investigate possible mechanisms behind this uncoupling of AVP signaling. The rats were examined 4 wk after CBL or sham operation. The CBL rats had increased plasma AVP concentrations (CBL: 3.2 ± 0.2 vs. sham: 1.4 ± 0.4 pg/ml, P < 0.05) and reduced AQP2 (0.62 ± 0.11) and phosphorylated AQP2 (0.50 ± 0.06) protein expression compared with sham-operated rats. However, examination of subcellular AQP2 localization by immunohistochemistry showed unchanged plasma membrane targeting in CBL rats, indicating a sustained ability of AQP2 short-term regulation. In a separate series of animals, thirsting was found to normalize AQP2 expression, indicating that AVP uncoupling in CBL rats is a physiological compensatory mechanism aimed at avoiding dilutional hyponatremia. Studies on microdissected collecting ducts from CBL rats showed decreased cAMP accumulation in response to AVP stimulation. The presence of the nonspecific phosphodiesterase inhibitor IBMX normalized the cAMP accumulation, indicating that cAMP-phosphodiesterase activity is increased in CBL rats. However, in contrast to this, Western blotting showed a decreased expression of several phosphodiesterase splice variants. We conclude that CBL rats develop an escape from AVP to prevent the formation of dilutional hyponatremia in response to increased plasma AVP concentrations. The mechanism behind AVP escape seems to involve decreased collecting duct sensitivity to AVP as a result of increased cAMP-phosphodiesterase activity. 9 B; z4 j% H/ I$ N; w
【关键词】 aquaporin phosphodiesterase common bile duct ligation kidney cAMP6 a8 z: N6 d- I- R
NORMALLY, THE KIDNEYS REGULATE urinary osmolality to compensate for variations in water intake and extrarenal water losses. Vasopressin (AVP) is released from the posterior pituitary gland in response to an increase in plasma osmolality or a decrease in intravascular volume. AVP regulates water permeability in the renal collecting duct (CD) by both short- and long-term regulation. The G s -coupled AVP type 2 receptors (V 2 receptor) are present in the basolateral membrane of the CD principal cells. V 2 receptor stimulation increases CD water permeability through cAMP-mediated protein kinase A (PKA) phosphorylation of aquaporin-2 (AQP2), whereby the water channels are shuttled from intracellular vesicles to the apical plasma membrane by exocytosis ( 30, 31, 36 ). For long-term regulation of body water, the total amount of AQP2 protein in the CD principal cells is increased along with AQP2 mRNA levels. This long-term regulation of AQP2 is controlled via PKA phosphorylation of the cAMP-responsive element binding protein (CREB), which stimulates AQP2 gene transcription ( 25, 41 ).: _* M8 F! M; s8 Y0 p9 A/ |( \
/ F! c5 Q8 y- n; h5 ?* _, SLiver cirrhosis and congestive heart failure (CHF) are frequent pathophysiological conditions, which in the late stages are associated with edema and dilutional hyponatermia due to increased plasma AVP concentrations. Several studies on common bile duct ligation (CBL)-induced liver cirrhosis have shown a decreased renal AQP2 expression despite increased plasma AVP concentrations ( 15, 20, 21 ). In addition, plasma sodium concentrations were shown to be normal. These findings suggest an uncoupling of the AVP effect on renal CD AQP2 regulation in CBL rats, preventing the development of hyponatremia despite increased plasma AVP levels. In contrast to these observations, CHF rats showed an increased AQP2 expression ( 32, 40 ), which indicates the absence of the otherwise appropriate mechanism of AVP uncoupling in CHF rats.
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" i/ p! ]6 U( H: W8 l3 [As hyponatremia induced by nonosmotic baroreceptor-mediated AVP release is known to be a predictor for mortality within advanced stages of liver cirrhosis and CHF ( 5, 6 ), it is of particular interest to study the phenomenon of AVP uncoupling. The aim of the present study was to investigate the mechanisms behind the regulation of AQP2 in rats with liver cirrhosis induced by CBL and characterized by increased plasma AVP concentrations. We confirmed the presence of AVP uncoupling within CBL rats by AQP2 Western blotting and plasma AVP measurements. In addition, we investigated the activation and localization of the AQP2 water channel by, respectively, analyzing the level of phosphorylated AQP2 (pAQP2), which is the active water-transporting form of AQP2, and the membrane targeting of AQP2 by immunohistochemistry.
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Measurements of cAMP accumulation in response to AVP stimulation within isolated, microdissected CDs were conducted to elucidate possible defects within the signaling from receptor to effector protein. Because cAMP is a key second messenger within this system and because phosphodiesterases (PDE) are the enzymes responsible for degradation of this molecule, it is possible that PDEs play a central role in the regulation of V 2 receptor signaling. Therefore, we also investigated the AVP-induced cAMP accumulation in the presence of the nonspecific PDE inhibitor IBMX. Furthermore, we conducted Western blotting on PDE isotypes 3 and 4 to support the results of the cAMP accumulation study.: O# N+ G" [' N, J3 s' w
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Finally, to investigate whether the observation of AVP uncoupling within CBL rats is truly the phenomenon of AVP escape, we subjected a series of CBL rats to thirsting. We analyzed the urine production and osmolality and whether thirsting was able to revert the decreased expression of AQP2.
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METHODS1 k; i; ^( V. K2 b" @& h" t4 g
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Female Wistar rats (230-250 g) from Charles River (Sulzfeld, Germany) were used for the experiments. The animals were housed in a temperature (22-24°C)- and moisture (40-70%)-controlled room with a 12:12-h light-dark cycle (lights on from 6 AM to 6 PM). Animals were given free access to tap water and a diet with 140 mmol/kg of sodium, 275 mmol/kg potassium, and 23% protein. All animal procedures followed the guidelines for the care and handling of laboratory animals established by the Danish government.2 G( A6 \) }9 h: i9 A- U5 e5 V
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Animal Preparation$ O5 r, V5 m* m {( c" c
; _4 Y' q" r7 P$ L+ ?Liver cirrhosis was induced by CBL as described by Kountouras and co-workers ( 24 ). Briefly, biliary obstruction induces portal inflammation and bile duct proliferation, which eventually results in the formation of cirrhosis. Control rats were subjected to sham operation.
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. }4 r+ Q7 s. e& |& SMeasurement of plasma AVP concentrations. Three weeks after CBL/sham operation, permanent medical grade Tygon catheters were implanted into the femoral artery as described previously ( 19, 35 ). Subsequently, the animals were housed individually. One week after instrumentation, arterial blood samples were drawn. To secure that the blood samples were collected during unstressed conditions, the rats were adapted to restraining cages by training for 2 h on 2 consecutive days. Blood samples (1 ml) were transferred to Transylol/EDTA tubes; plasma was isolated by centrifugation at 4,000 g and stored at -20°C until analysis. AVP was extracted from plasma on C-18 SEP-Pak cartridges and measured by RIA as previously described ( 23 )." ?! j. r8 M" S- I# R
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Isolation of CDs. After blood sample collection, the rats were anesthetized by halothane in N 2 O-O 2 (2:1) and a ligature was placed around the aorta between the take-off of the right and left renal artery. The aorta was cannulated and the left kidney was perfused with 25 ml of ice-cold digestion solution containing 0.2 U/ml collagenase A (0.22 U/mg; Boehringer Mannheim), 1 mg/ml BSA, and O 2 -aerated microdissection solution (135 mM NaCl, 1.0 mM Na 2 HPO 4, 2 H 2 O, 5.0 mM KCl, 1.2 mM MgSO 4, 2.0 mM CaCl 2, 1.2 mM NaSO 4, 5.0 mM HEPES, and 5.5 mM D -glucose monohydrate, pH 7.4). After perfusion, the left kidney was removed and placed in ice-cold microdissection solution. The kidney was sliced twice longitudinally and the central slice (thickness of 2 mm) was divided in two. Wedge-shaped pieces were cut from each of the two halves, and from these cortex and the inner stripe of outer medulla (ISOM) were isolated and placed in glass tubes containing ice-cold microdissection solution. The microdissection solution was exchanged with preheated digestion solution and the samples were incubated in a 37°C shaking water bath under O 2 aeration. The incubation time was 24-25 min depending on the renal zone to be dissected. After incubation, the tissue was rinsed three times in ice-cold microdissection solution and the CDs from the cortex and ISOM were isolated by microdissection under a stereomicroscope. The microscope was connected to a camera and a computer, which made it possible to measure the length of the isolated CDs by use of the Olympus OlyLite 2.0 software. Microdissection was performed at 4°C in a dissection dish placed on a water-cooled transparent chamber with light transmitted directly through the chamber from beneath. This arrangement enabled the identification and isolation of CDs from other tubular segments.
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AVP-mediated cAMP accumulation in isolated CDs. The isolated CDs were transferred to coverslips by use of BSA-coated pipette tips. The microdissection solution in which the tubules were transferred was removed using filter paper and 2.5 µl of incubation media (Krebs Ringer buffer) containing 125 mM NaCl, 250 mM urea, 5 mM KCl, 1.2 mM MgSO 4, 7 H 2 O, 10 mM sodium acetate, 10 mM D -glucose monohydrat, 20 mM Tris base, 2 mM Na 2 HPO 4, 2 H 2 O, 0.8 mM CaCl 2, 2 H 2 O was added to the samples (the osmolarity of the buffer should be 280 mosM). For basal CD cAMP accumulation, 10-12 mm of CD were incubated for 20 min in a 37°C water bath after which the samples were immediately placed on dry ice and stored at -80°C for later measurement of cAMP. AVP-stimulated CD cAMP accumulation was measured after 20-min incubation at 30°C in incubation media containing one of the following AVP concentrations: 10 -6 M (3- to 4-mm CD), 10 -8 M (5- to 6-mm CD), 10 -9 M (8- to 9-mm CD), and 10 -10 M (9- to 10-mm CD).
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9 w) U) v2 t1 n$ G4 KFinally, 1.5- to 2-mm CD was incubated in a medium containing AVP 10 -6 and 5·10 -4 M of the nonspecific PDE inhibitor IBMX.
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1 R% Y" ?8 k9 ]0 sTo ensure correct pH in the incubation media, the solutions were aerated with 95% O 2 -5% CO 2 and the pH was measured just before using the media.& o' T* O: f3 N* b3 f
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Measurement of cAMP in the samples was conducted by use of a commercial cAMP Enzyme Immunoassay kit from Cayman Chemicals (cat. no. 581001). All cAMP concentrations were expressed relative to the length of the CDs within the individual samples.' |$ [0 Z, C) e
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Western blotting. The right kidney was removed, immediately frozen in liquid nitrogen, and stored at -80°C until processing for membrane fractionation. Whole kidneys were homogenized using a tissue homogenizer (Ultra-Turrax T8, Ika, Staufen, Germany) in a 9-ml ice-cold homogenizing buffer containing 300 mM sucrose, 25 mM imidazol, 1 mM EDTA-disodium salt, and the following protease inhibitors: Pefabloc 0.1 mg/ml buffer and leupeptin 4 µg/ml buffer; and phosphatase inhibitors: sodium ortho-vanadate 184 µg/ml buffer, sodium fluoride 1.05 mg/ml buffer, and okadeic acid 82 ng/ml buffer; pH was adjusted to 7.2 with 0.1 M HCl.
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After centrifugation (4,000 g, 4°C, 15 min), the supernatant was isolated and the protein concentration was measured by use of a commercial kit (Pierce BCA Protein Assay Reagent Kit cat. no. 23226, Pierce, Rockford, IL). All samples were diluted to a final protein concentration of 1 µg/µl adding sample buffer (in the final solution: 486 mM Tris·HCl, pH 6.8, 8.7% glycerol, 104 mM SDS, 0.0875 mM bromphenolblue), dithiothreitol (25 mM in the final solution), and homogenizing buffer. Finally, the samples were solubilized at 90°C for 10 min.4 |0 b' J7 S0 p f
; A) ?4 M% X/ U3 `% b1 \; hThe samples were run on 12% polyacrylamide gels and the proteins were electrotransferred to nitrocellulose membranes (60 min, 100 V, 200 mA). After unspecific binding sites were blocked (60 min in PBS-T buffer containing 5% milk), the membranes were probed overnight at 4°C with the appropriate primary antibody. For measurement of AQP2, we used a rabbit polyclonal anti-AQP2 antibody raised against rat AQP2 (LL127) ( 31 ). For measurement of pAQP2, we used a rabbit polyclonal antibody, which only recognizes AQP2 phosphorylated at serine 256 (AN244) ( 7 ).
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" G1 z# }! g) z5 L. t/ _The labeling was visualized with horseradish peroxidase (HRP)-conjugated secondary antibody (P448; Dako, Glostrup, Denmark) using an enhanced chemiluminescence system (ECL , Amersham). The 29- and 35- to 50-kDa bands, corresponding to nonglycosylated AQP2 and glycosylated AQP2, respectively, were scanned by the FluorX Max2 multiImager (Bio-Rad Laboratories). Densitometry of individual bands was quantitated using the software program Quantity One, version 4.2.3 (Bio-Rad Laboratories).
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Immunohistochemisty. Four weeks after CBL or sham operation, another series of animals ( n = 5 in both groups) was anesthetized by isoflurane in N 2 O/O 2 (2:1) and both kidneys were perfusion-fixed in 3% paraformaldehyde in 0.1 M sodium cacodylate buffer. After 1 h of postfixation (perfusion solution), the central cone of the kidneys was dissected, rinsed, and stored in cacodylate buffer at 4°C until paraffin embedding 24 h later. Two-micrometer sections of the in vivo perfusion-fixed kidneys were incubated overnight at 4°C with a polyclonal rabbit anti-AQP2 antibody (LL127). Sections were then incubated with goat anti-rabbit Alexa 488 (Molecular Probes, Eugene, OR) for 60 min at room temperature. After being rinsed with PBS, sections were mounted in glycerol mounting medium and examined using a Leica TCS SP2 laser confocal microscope ( 13, 14 ). The ratio between membrane-bound and subapical AQP2 was quantified by mean pixel intensity [intensity units (IU)] in an 8 x 8-pixel area of the plasma membrane and the subapical cytosol using the software program Quantity One, version 4.2.3 (Bio-Rad Laboratories). Membrane localization was defined as the apical 4 x 8-pixel part of the 8 x 8-pixel area. Membrane localization of AQP2 was then expressed as a fraction of the total intensity. For each sample, a total number of six analyses were performed.) \9 ^* o, U w1 I3 N/ G0 |
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& f8 k; V4 k9 s3 W) ~Western blotting of PDE. Four weeks after CBL or sham operation, rats were anesthetized by isoflurane in N 2 O/O 2 (2:1) and both kidneys were rapidly removed, frozen in liquid nitrogen, and stored at -80°C until processing for membrane fractionation.1 Z0 k( G4 h' g7 `' Q
* C( w1 z! ~$ }; c+ T7 Z) X3 `! \Sample preparation and blotting procedures were conducted as described in series 1 except that the kidneys were homogenized in 6 ml buffer, the sample protein concentration was 5 µg protein/µl, the proteins were blotted to PVDF membranes, and when using a primary goat antibody, the blots were blocked for 1 h in PBS-T buffer containing 0.5% Tween 20 and the secondary antibody used was a HRP-conjugated rabbit anti-goat antibody (P449, Dako).
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We focused on PDE3 and 4, which are the main PDE gene families present in the renal collecting ducts ( 7a, 42 ). The PDE3 gene family includes the PDE3A and B subtypes ( 24a ). The PDE4 gene family includes the PDE4A, B, C, and D subtypes ( 7a ), which furthermore includes several different isoforms. We conducted Western blotting on the PDE3B subtype and the PDE4A, B, and D subtypes.0 u' U) |( Q1 j/ W
2 b g- ^( }. \. Q" HFor the measurement of PDE3B expression, we used a commercially available affinity-purified goat anti-PDE3B isoform-specific antibody (no. CYAB222, Cytomyx), which recognizes a 135-kDa band. For the measurement of PDE4A expression, we used an affinity-purified rabbit anti-PDE4 isoform-specific antibody (no. CYAB230, Cytomyx), which recognizes the following: a 66 (PDE4A1)-, 76 (PDE4A?)-, 102 (PDE4Ax)-, 10 (PDE4A8)-, and 109 (PDE4A5)-kDa band. For measurement of PDE4B expression, we used an affinity-purified rabbit anti-PDE4B isoform-specific antibody (no. CYAB245, Cytomyx), which recognizes the following: a 66 (PDE4B4)-, 78 (PDE4B2)-, 100 (PDE4B3)-, and 107 (PDE4B1)-kDa band. Finally, for the measurement of PDE4D expression, we used an affinity-purified rabbit anti-PDE4D isoform-specific antibody (no. CYAB255, Cytomyx), which recognizes the following: a 68 (PDE4D1 and 2)-, 95 (PDE4D3)-, 105 (PDE4D5)-, and 119 (PDE4D4)-kDa band.
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We obtained specific binding for the following PDE isoforms: PDE4B4 (66 kDa), PDE4D1 and 2 (68 kDa), and PDE4D4 (119 kDa).4 i. e$ L3 c, H+ ]; O2 ~( h% W
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Series 4
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, u* o+ G4 A5 e9 C2 {- L1 ?. aMeasurement of urine volume and urine osmolality of thirsted CBL rats. Four weeks after the initial CBL or sham operation, the rats were placed in metabolic cages and urine collections were made for the measurement of urine volume and osmolality. After a 2-day adaptation period, 24-h urine production was collected for 5 consecutive days consisting of a 3-day baseline period with free access to standard rat chow and water and a 2-day thirsting period. Urine volume was determined gravimetrically and urine osmolality was analyzed by use of a cryomatic osmometer (model 3 CII, Advanced Instruments, Needham Heights, MA).2 J2 o* ^( r. @* [7 g5 J. j H: {# O
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Western blotting. After the thirsting period, the kidneys were removed and stored at -80°C until sample preparation. Sample preparation and blotting procedures were conducted as described in series 3 and the following proteins were analyzed: AQP2 (Santa Cruz, no. sc-9882), PDE3B, and PDE4A, B, and D.
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Statistics; W7 P9 z8 J: R, O1 W9 m6 A# p
$ a3 R5 P" S4 i! Z/ pData are presented as means ± SE. The effect of increasing concentrations of AVP on CD cAMP accumulation (dose-response studies) was analyzed by two-way analysis of variance. All other comparisons were analyzed with Student's unpaired t -test. Differences were considered significant at probability levels P of 0.05; n indicates the number of animals.
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RESULTS$ _: \# N1 A9 Z
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As previously shown ( 15, 17, 21 ), cirrhotic rats had significantly increased plasma AVP concentrations [CBL: 3.2 ± 0.2 pg/ml ( n = 15) vs. sham: 1.4 ± 0.4 pg/ml ( n = 12); P 1 Q3 T7 f* Y" R& G( s+ N3 o
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Fig. 1. Plasma vasopressin concentrations (pAVP; A ) and Western blots of whole kidney membrane preparations (7 µg protein/lane) from rats subjected to common bile duct ligation (CBL) or sham operation (Sham) 4 wk earlier. The Western blots were incubated with anti-aquaporin-2 (AQP2; B ) or anti-phosphorylated AQP2 (pAQP2; D ) antibody and reveal a 29- and a 35- to 50-kDa band corresponding to nonglycosylated and glycosylated protein, respectively. Densitometry ( C and E ) was performed on all samples and mean density of the sham-operated rats was normalized to 1.0. Values are means ± SE. * P
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AQP2 and pAQP2 Protein Levels
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. x% V$ u2 x! P$ U' ~, sFigure 1 B shows a Western blot of whole kidney membrane fractions (7 µg protein/lane). The affinity-purified anti-AQP2 protein antibody (LL127) recognizes a 29- and a 35- to 50-kDa band, corresponding to the nonglycosylated and glycosylated AQP2 protein, respectively. Densitometry of all samples ( Fig. 1 C ) confirmed previous findings ( 15, 17, 20, 21 ) by showing a significantly decreased AQP2 protein level in the cirrhotic rats (0.62 ± 0.11 of the sham rats; P + B! F! z6 C5 z/ d3 h
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Immunohistochemical Visualization of AQP2 in the CDs
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To investigate whether cirrhotic rats displayed changes in the subcellular distribution of AQP2 within the CD principal cells, we conducted immunohistochemistry with the specific anti-AQP2 antibody LL127. Overall, there were no changes in the fraction of AQP2 associated with the plasma membrane in either of the renal zones in the cirrhotic rats compared with sham-operated controls [cortex: sham: 66 ± 2% ( n = 5) vs. CBL: 67 ± 2% ( n = 5); P = 0.72; outer medulla: sham: 62 ± 2% ( n = 5) vs. CBL: 66 ± 3% ( n = 5); P = 0.22; inner medulla: sham: 59 ± 3% ( n = 5) vs. CBL: 66 ± 2% ( n = 5); P = 0.13; Fig. 2 ]. This suggests that despite increased circulating levels of AVP in CBL rats, there was no evidence of increased apical targeting of AQP2.
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Fig. 2. Localization of AQP2 in cortical, outer, and inner medullary collecting ducts from rats subjected to CBL or Sham 4 wk earlier. The kidneys were perfusion-fixed in vivo and paraffin-embedded. Two-micrometer-thin sections were incubated overnight at 4°C with a polyclonal rabbit anti-AQP2 antibody (LL127) and then incubated with goat anti-rabbit Alexa 488 (Molecular Probes) antibody for 60 min at room temperature. Sections were examined by laser confocal microscopy. The ratio between membrane-bound and subapical AQP2 was quantified by mean pixel intensity in an area of plasma membrane and supapical cytosol. Membrane localization was defined as the apical half of the pixel area. Membrane localization of AQP2 was expressed as a fraction of the total intensity. The membrane fraction of AQP2 was unchanged in all renal zones compared with controls (for exact values, see RESULTS ).% Y, A+ n# V. I* p s
8 l, K- [3 b2 v3 l; k0 l+ E* [Thirsting of CBL Rats7 O6 q1 o& [1 u: Q: y
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To address the question of whether the uncoupling of V 2 receptor signaling within CBL rats is a physiological compensatory mechanism, rather than a toxic effect on the CDs, we investigated the effect of thirsting on water handling and AQP2 expression in CBL and sham rats, respectively. We found that the effects of thirsting were the same in both the CBL and sham group, showing a simultaneously significant decrease in urine volume and increase in urine osmolality ( Fig. 3, A and B ). Moreover, measurement of whole kidney AQP2 expression showed that 48-h thirsting eliminated the difference in AQP2 protein expression observed between CBL and sham rats ( Fig. 3, C and D ). Thus, because thirsting increased renal AQP2 protein levels in CBL rats to the same level as in sham-operated controls, these results suggest that decreased renal AQP2 expression in CBL rats is due to a physiological adaptation rather than an unspecific toxic effect on the CDs.
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, T( l$ c5 z6 M5 w6 g% ~% L% xFig. 3. Urine production, urine osmolality ( A and B, respectively), and whole kidney AQP2 expression ( C and D ) in response to thirsting of rats subjected to CBL or Sham operation 4 wk earlier. A and B : rats were placed in metabolic cages. After a 2-day adaptation period, 24-h urine production was collected for 5 consecutive days: a 3-day baseline period and a 2-day thirsting period. Urine volume ( A ) and urine osmolality ( B ) were measured. Both the CBL and Sham rats showed a simultaneous decrease in urine volume and increase in urine osmolality on thirsting. C : AQP2 Western blotting was performed on whole kidney membrane fractions using the anti-AQP2 antibody sc-9882 (Santa Cruz Biotechnology). The antibody revealed a 29- and a 35- to 50-kDa band corresponding to nonglycosylated and glycosylated AQP2 protein, respectively. D : densitometry was performed on all samples and the mean density of the Sham rats was normalized to 1.0. Values are means ± SE; n = 6 in both groups.
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AVP-Induced cAMP Accumulation" L6 }9 Z5 W4 i
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It is well described that AVP through activation of V 2 receptors stimulates cAMP generation in the CDs. We therefore conducted dose-response studies of AVP-induced cAMP accumulation in isolated CDs to determine if cirrhosis was associated with changes in the AVP-mediated cAMP accumulation. Because we previously showed that downregulation of AQP2 was most pronounced in the cortex and outer medulla ( 20 ), we restricted our examination to CDs from these zones. The accumulation of cAMP in response to 20 min of AVP stimulation was significantly attenuated in the cirrhotic rats compared with sham-operated controls in both kidney zones ( Fig. 4 ). To examine whether this impaired response to AVP could be caused by changes in the activity of the PDE, which are the enzymes responsible for the degradation of cAMP, we measured the cAMP accumulation in response to AVP stimulation in the presence of the nonspecific PDE inhibitor IBMX. The presence of IBMX normalized the cAMP accumulation in isolated CDs from cirrhotic rats ( Fig. 5 ).
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Fig. 4. Dose-response curves of AVP (10 -10 to 10 -6 M)-mediated cAMP accumulation in isolated cortical (CCD) and outer medullary (OMCD) collecting ducts from rats subjected to CBL or Sham 4 wk earlier. Isolated collecting ducts were incubated with increasing concentrations of AVP for 20 min at 30°C. The cAMP accumulation was measured using an enzyme-linked immunoassay and expressed as cAMP concentration per millimeter of collecting duct. Values are means ± SE. conc, Concentration. * P
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) H4 K) C1 n0 _( N8 p0 {+ B1 RFig. 5. cAMP accumulation after 20-min AVP 10 -6 M incubation at 30°C with or without the unspecific phophodiesterase inhibitor IMBX (4·10 -5 M) in isolated CCD and OMCD from rats subjected to CBL (filled bars) or Sham (open bars) 4 wk earlier. The cAMP accumulations were measured using an enzyme-linked immuno assay and expressed as cAMP concentration per millimeter of collecting duct. Values are means ± SE. * P : t1 p' U! q. @5 e
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Western Blotting of PDE3B and PDE4A, B, and D Subtypes1 G: ?0 B1 c! f3 Q' {) W/ d
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To investigate the role of PDEs in the AVP uncoupling in CBL rats further, we analyzed the renal expression of PDE subtypes 3B, 4A, 4B, and 4D by Western blotting.
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Measuring the PDE4B expression within whole kidney homogenates, we found the 66-kDa PDE4B4 isoform to be significantly decreased within CBL rats [sham: 1 ± 0.07 ( n = 6) vs. CBL: 0.59 ± 0.09 ( n = 5); P
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Fig. 6. Western blotting of whole kidney PDE4B expression. Conducting Western blotting on the PDE4B isoform a single 66-kDa band appeared, corresponding to the PDE4B4 splice variant. A : rats with CBL showed a marked decrease within PDE4B4 expression compared with Sham rats. B : same level of downregulation was observed comparing the Sham thirst group with the CBL thirst group. Analyzing the effect of thirsting on, respectively, Sham rats ( C ) and CBL rats ( D ) a further decrease was found within the expression of the PDE4B4 splice variant. For each blot, densitometry was performed on all samples and the mean density of the control group was normalized to 1.0. Values are means ± SE. * P
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6 N1 \; S; }% G' G! TAnalyzing the expression of the 119-kDa PDE4D4 splice variant, we found the same pattern of regulation as observed for the PDE4B isoform, showing decreased expression comparing sham and CBL [sham: 1 ± 0.05 ( n = 6) vs. CBL: 0.60 ± 0.11 ( n = 5); P 9 p% f1 z8 ?- F- l5 I3 m% p$ J$ V
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Fig. 7. Western blotting of whole kidney PDE4D expression. The blots revealed bands at 68-kDa (PDE4D1 and D2) and 119-kDa (PDE4D4). Only the 119-kDa PDE4D4 band showed difference within expression levels in a comparison of rats subjected to CBL or Sham operation ( A ) and Sham thirst to CBL thirst ( B ), respectively. For each blot, densitometry was performed on all samples and mean density of the control group was normalized to 1.0. Values are means ± SE. * P 4 f9 f( o/ v9 Q& q. N; e$ | d
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Analyzing both the PDE3B and PDE4A expression, we did not obtain any specific binding and it was therefore not possible to draw any conclusions in regard to these PDE subtypes.
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The present study confirms previous findings of increased plasma AVP levels and decreased renal expression of the AQP2 water channel and thereby the presence of AVP uncoupling in rats with CBL-induced liver cirrhosis ( 11, 15, 20, 21 ). Furthermore, this study shows that the downregulation of AQP2 is associated with a decrease in the AVP-induced cAMP accumulation of isolated CDs from CBL rats. Moreover, Western blotting of pAQP2 and immunohistochemical studies of AQP2 localization indicate that targeting of the AQP2 water channel remains unchanged in the CBL rats, supporting the conclusion of uncoupling of AVP signaling within CBL rats.' C1 A3 Z1 g& |' i' k' W! |
2 o! E7 z8 M7 J `& m7 YIf decreased renal AQP2 protein expression despite increased plasma AVP levels in CBL-induced liver cirrhosis is a physiological compensatory mechanism aimed to avoid dilutional hyponatremia, then it would be expected that thirsting increased AQP2 expression in CBL rats to the same level as in sham-operated control rats. In fact, we found that urine production and AQP2 expression were the same in thirsted CBL and sham rats. Thus the uncoupling of AVP signaling within CBL rats is a physiological rather than a toxic mechanism. In conclusion, this study indicates that cirrhosis induced by CBL is associated with a physiological escape from AVP in the CDs.
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. C2 W) I; S$ w! [9 pA number of clinical conditions are associated with increased plasma AVP concentrations. Severe CHF, late stages of liver cirrhosis, and severe nephrotic syndrome are all associated with increased AVP secretion, which most probably is baroreceptor mediated ( 3, 22, 37, 40 ). Moreover, a number of different conditions including neoplasia, neurological disorders, and drug treatment can induce the syndrome of inappropriate antidiuretic hormone secretion, which is characterized by some degree of hyponatremia and plasma hypotonicity and negative free water clearance ( 28 ). However, plasma sodium concentrations rarely fall below 120 mM despite the presence of even very high plasma concentrations of AVP, indicating an escape from the action of AVP on the CDs. Until recently, little was known about the mechanisms involved in the AVP-escape phenomenon. However, Ecelbarger and co-workers ( 9 ) intensively investigated the AVP-escape phenomenon in an animal model where AVP escape was induced by combined water loading and V 2 receptor agonist (dDAVP) treatment. In this model, AVP escape seems to be associated with downregulation of V 2 receptor binding and decreased cellular cAMP accumulation along with decreased AQP2 protein and mRNA levels ( 8, 39 ).
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, g% q J/ @. Q4 G7 Q4 nThe AVP-escape phenomenon is of particular interest in relation to advanced stages of liver cirrhosis and CHF because the presence of hyponatremia is an independent predictor for mortality ( 5, 6 ). Schrier and co-workers ( 40 ) as well as our group ( 32 ) examined the role of AQP2 protein levels and subcellular localization in rats with severe CHF associated with hyponatremia and increased plasma AVP levels. These studies showed that renal AQP2 mRNA and protein levels were significantly increased and associated with a marked increase in plasma membrane targeting of the water channel. Recent followup studies from our laboratory showed that the aquaretic response to selective V 2 receptor blockade is increased in CHF rats with normal plasma AVP levels ( 38 ), indicating increased AVP-mediated tubular water reabsorption. Furthermore, we showed that AQP2 protein levels and the amount of phosphorylated and thereby plasma membrane-associated AQP2 are increased in CHF rats with normal plasma AVP concentration ( 13 ). These findings strongly indicate a lack of AVP escape in the CDs of CHF rats.5 `) h! C" i5 M$ `% {
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In contrast to these findings in CHF rats, most studies including the present show that rats with liver cirrhosis induced by CBL exhibit downregulation of the renal AQP2 protein in the presence of increased plasma AVP levels and normal plasma sodium concentrations ( 11, 15, 20, 21 ). Regarding liver cirrhosis, several conflicting data on the AQP2 expression have been obtained. The initial reports from two Japanese groups showed increased AQP2 expression in rats with severe decompensated cirrhosis induced by CCl 4 treatment ( 4, 12 ). It could be asked whether the discrepancies between these findings within the CCl 4 model and the CBL model are due to marked differences in the pathophysiology and whether one of the models is more representative for human cirrhosis than the other. A number of findings argue against the possibility that the renal patophysiological changes are different in the two models of cirrhosis. Based on detailed studies on the time course of the development of renal dysfunction in CCl 4 rats, we recently showed that renal function during the compensated state of the disease was comparable to the changes in the CBL model ( 22 ). Furthermore, at least two studies including one from our own laboratory showed that AQP2 expression is unchanged in rats with more severe, decompensated cirrhosis induced by CCl 4 treatment despite significantly elevated plasma AVP concentrations ( 10, 16 ). However, this does not explain why the initial studies published in 1995 by the Japanese groups ( 4, 12 ) showed increased AQP2 protein levels in CCl 4 -induced cirrhosis. One possible explanation although could be that these studies were made in rats with very severe water disturbances in the terminal state of the disease. Unfortunately, no information about renal function in terms of renal perfusion, glomerular filtration rate, or segmental tubular function was reported in the two Japanese studies.
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1 ^' ^. y. M& L5 ~5 v$ @The present findings strongly indicate that rats with liver cirrhosis at least in the compensated and "early" decompensated stages develop an escape from AVP-induced antidiuresis, which can be viewed as an adaptive mechanism limiting the degree of hyponatremia and thereby protecting the body against fatal hyposmolality. Our working hypothesis on the AVP escape and liver cirrhosis has therefore been that AVP escape is observed in the compensated and "early" decompensated stage of liver cirrhosis obtained by CBL. In the late decompensated stage (associated with hyponatremia and ascites formation), it is no longer possible to maintain this otherwise appropriate escape mechanism preventing development of the fatal hyponatremia. This shift from presence to lack of AVP escape is indicated by a shift in AQP2 expression from being decreased to being increased compared with controls.
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( A0 |% e* [4 \3 e7 u- yThe present study furthermore showed that the AQP2 downregulation is associated with decreased AVP-induced cAMP accumulation in the CDs. However, despite decreased AQP2 levels in the CBL rats, substantial amounts of pAQP2 were still present and immunohistochemical quantifications showed that the fraction of AQP2 present within the apical membrane was unchanged in the CBL rats. This suggests that despite uncoupling of the AVP signaling within the CDs, short-term regulation of the AQP2 water channel was still present within the CBL rats.. d0 |. W, O" H2 i6 u) \9 X! h
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This study showed that the AVP escape of CBL-induced liver cirrhosis is associated with decreased cAMP accumulation within the CDs. In addition, we found that the presence of the unspecific PDE inhibitor IBMX normalized the AVP-induced cAMP accumulation in the CDs of CBL rats, suggesting that increased PDE activity plays a part in AVP escape.- R7 H, i- K8 ]* H
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It has previously been shown that cirrhotic rats display increased activity of renal cGMP PDEs associated with a marked impaired natriuretic effect of ANP ( 2, 29 ). A mechanism that must be regarded as inappropriate, because it most probably will induce sodium retention. However, the mechanisms behind both the inappropriately increased cGMP PDE activity as well as the presumable appropriately increased cAMP PDE activity in cirrhosis remain to be explained.2 d& n+ m1 w1 l4 c8 w
- y0 G- \2 Q% \6 e' B. r9 T. x, ?We pursued the topic of increased cAMP PDE activity by measuring the protein expression of several cAMP PDE subtypes in whole kidney homogenates from CBL rats. If increased CD PDE activity is truly the mechanism behind the AVP escape observed in CBL-induced liver cirrhosis, increased PDE protein expression would be an expected finding. However, our studies showed a decreased expression of the PDE4B4 and the PDE4D4 isoforms, whereas no difference was observed for the PDE4D1 and 2 isoforms in a comparison of CBL and sham-operated control rats. Furthermore, our studies showed that thirsting induced a further decrease of PDE4B4 but not PDE4D4 expression. Evaluating these data, it could be argued that the increased CD PDE activity in CBL rats must be a result of increased enzymatic activity of the single PDE molecule and not due to an increased number of PDE molecules. The present data are unable to give a more precise answer to this disconnect between enzymatic activity and expression levels of PDEs. Further studies including time course studies of PDE activity and expression levels in CBL rats are warranted.
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It can be questioned why CBL rats, despite decreased AQP2 water channel expression, have normal urine production. We previously showed that CBL rats have increased Na-K-2Cl cotransporter (NKCC2) expression ( 15 ), suggesting an increased Na reabsorption capacity in the thick ascending limb of the loop of Henle's (TAL). This would result in an increased corticomedullary osmotic gradient, which would increase the driving force for water reabsorption. Interestingly, not only liver cirrhosis seems to be associated with altered TAL function. Increased NKCC2 expression has also been found in rats with CHF ( 27, 33, 38 ), sepsis-induced acute renal failure ( 18 ), and in animal models of hypertension ( 1, 26 ). Together, these data seem to support a role of NKCC2 regulation in a number of pathophysiological conditions with impaired renal sodium handling." H t) B& o$ T; p% f
3 v% T0 i2 m% oFinally, it could be questioned whether the present study showing that rats with CBL-induced cirrhosis develop an escape from AVP-induced antidiuresis have any clinical implications? A recent study by Pedersen and co-workers ( 34 ) showed that the urinary excretion of AQP2 was significantly attenuated in cirrhotic patients, whereas the urinary AQP2 excretion was significantly increased in CHF patients compared with control persons. This finding indicates the presence of AVP escape in cirrhotic patients and the absence of AVP escape in CHF patients as observed in our animal models of cirrhosis and CHF. Further studies of the mechanism of AVP escape in conditions with extracellular volume expansion are therefore warranted.# j* l4 \+ ^! U
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GRANTS
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This work received financial support from The Danish Medical Research Council, The Novo Nordic Foundation, The Eva and Robert Voss Hansen Foundation, The Ruth Kønig-Petersen Foundation, The Knud Øster-Jørgensen Foundation, The Helen and Ejnar Bjørnow Foundation, and the Aage Thuesen Bruun Foundation.
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ACKNOWLEDGMENTS
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0 s U( M" t% UThe technical assistance of I. Nielsen, B. Seider, L. Frandsen, A. Nielsen, and I. M. Jalk is acknowledged. We gratefully acknowledge Dr. J. Warberg for performing the plasma AVP analyses.2 H) j# o) {0 I5 I( y
【参考文献】, @2 u& U+ ^' m3 h) G) ?8 p
Alvarez-Guerra M and Garay RP. Renal Na-K-Cl cotransporter NKCC2 in Dahl salt-sensitive rats. J Hypertens 20: 721-727, 2002.
. n3 y A/ {) x0 ~2 ^% [3 t; r2 y8 z7 p/ [( Q; ^* o
" c' ]3 H5 Q8 {8 e! G
, n6 l/ c' w9 H* M$ q$ w' N5 NAngeli P, Jimenez W, Veggian R, Fasolato S, Volpin R, MacHenzie HS, Craighero R, Libera VD, Sticca A, Arroyo V, and Gatta A. Increased activity of guanosine 3'-5'-cyclic monophosphate phosphodiesterase in the renal tissue of cirrhotic rats with ascites. Hepatology 31: 304-310, 2000.5 T$ Y1 ^" b; n( m4 X3 Q
n% N# P3 P/ | ]; C$ w$ m4 v( C! z
: c" y+ E [/ j/ M5 E* m
; M5 W' l% E. J2 ~' H1 ZApostol 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.
S/ \+ Z! r) r& R* v8 L/ K7 h! w* i/ K9 T3 W
& F/ j/ {/ I4 F6 _" f( B3 |
4 c, g6 z1 w$ W- DAsahina Y, Izumi N, Enomoto N, Sasaki S, Fushimi K, Marumo F, and Sato C. Increased gene expression of water channel in cirrhotic rat kidneys. Hepatology 21: 169-173, 1995.
+ S* c; u5 p6 s+ w9 W2 s8 v$ Q2 ]2 f3 y+ x4 j0 n5 ^. v
' w( L) j+ ^; M7 L! J0 a
! I K( _: _: T' a4 n5 eBorroni G, Maggi A, Sangiovanni A, Cazzaniga M, and Salerno F. Clinical relevance of hyponatraemia for the hospital outcome of cirrhotic patients. Dig Liver Dis 32: 605-610, 2000.
+ T! k9 }" w. g5 K0 q$ O) o; w. e0 D4 ?& L8 \
/ B6 I2 }( {6 s, d5 j' M* [! z( i& v" \* c/ A. |! q& S1 b8 N
Brophy JM, Deslauriers G, and Rouleau JL. Long-term prognosis of patients presenting to the emergency room with decompensated congestive heart failure. Can J Cardiol 10: 543-547, 1994.
) w) W3 h4 k' G. M2 n
0 ]1 H2 K8 J5 C, O7 J. p
: |( N0 ] ~2 L, B
0 i: w0 K* a' v) oChristensen BM, Zelenina M, Aperia A, and Nielsen S. Localization and regulation of PKA-phosphorylated AQP2 in response to V 2 -receptor agonist/antagonist treatment. Am J Physiol Renal Physiol 278: F29-F42, 2000.
- l5 T/ g8 C6 @2 D
, h8 u: j3 x; ?4 \4 z5 O5 c
5 j8 a% E! u, C& P9 ~$ ]6 j
2 [ [# M: o& C+ S' a- X. N# N1 }Dousa T. Cyclic-3',5'-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney. Kidney Int 55: 29-62, 1999.
5 f U! ^6 Q* e( @1 E7 ]4 T. i1 F3 ^* Z: r1 n- F6 ~1 q9 ~; r8 M
) ^" U" G$ V7 X" z2 H$ n0 _6 E. ~ {$ f$ p9 j" E
Ecelbarger CA, Chou CL, Lee AJ, DiGiovanni SR, Verbalis JG, and Knepper MA. Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct. Am J Physiol Renal Physiol 274: F1161-F1166, 1998.! m) D2 c7 y- S- \ u- k: W r; C
/ e g$ `( ~' f. M* \& Z$ \- L4 Z) q" g
% a+ K1 {5 ]1 P3 {$ nEcelbarger CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, and Verbalis JG. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 99: 1852-1863, 1997.% x' Q7 r1 [; ]0 }2 e
7 S2 ~ B2 `/ M3 i8 s
4 E9 L. G+ v' c4 d. {, ^0 i1 X0 [7 n1 u' O1 z9 C6 h- Z0 A
Fernández-Llama P, Jimenez W, Bosch-Marce M, Arroyo V, Nielsen S, and Knepper MA. Dysregulation of renal aquaporins and Na-Cl cotransporter in CCl 4 -induced cirrhosis. Kidney Int 58: 216-228, 2000.0 s. n. L$ t- I/ e/ g
, L# T n6 p- C: x2 U
" C, A w5 i" q& V" ~- x" T" f
3 t! }6 i4 L" R/ _Fernández-Llama P, Turner R, Dibona G, and Knepper MA. Renal expression of aquaporins in liver cirrhosis induced by chronic common bile duct ligation in rats. J Am Soc Nephrol 10: 1950-1957, 1999.2 b0 D5 K( {4 p1 G+ V7 d
2 }0 [# \. q/ P$ R1 A* |
! `% i' e/ T1 l1 Q
; h9 o4 v9 e0 r4 i7 J) s- r4 I Z$ TFujita N, Ishikawa SE, Sasaki S, Fujisawa G, Fushimi K, Marumo F, and Saito T. Role of water channel AQP-CD in water retention in SIADH cirrhotic rats. Am J Physiol Renal Fluid Electrolyte Physiol 269: F926-F931, 1995.
) `) p) E$ o+ ~' N% A J# N: z
6 x! f" M# M& {& ?' B) d* i" P$ e! ]4 C
' T( w8 U3 n: z) P& {5 T) K4 hHadrup N, Petersen JS, Praetorius J, Meier E, Græbe M, Brønd L, Staahltoft D, Nielsen S, Christensen S, and Jonassen TEN. Opioid receptor-like 1 stimulation in the collecting duct induces aquaresis through vasopressin-independent aquaporin-2 downregulation. Am J Physiol Renal Physiol 287: F160-F168, 2004.
( f! c" o. z& w) x5 u |
3 [: {; W+ [0 P4 R6 F* I9 o4 z- y" _6 A! h3 S3 f
1 ?5 e7 M6 Y3 k& Z+ u) O! _Hager H, Kwon TH, Vinnikova AK, Masilamani S, Brooks HL, Frokiaer J, Knepper MA, and Nielsen S. Immunocytochemical and immunoelectron microscopic localization of -, -, and -ENaC in rat kidney. Am J Physiol Renal Physiol 280: F1093-F1106, 2001.. t, h! w( t% P- s3 ?
" A6 y3 h G9 B
. O0 M; Q' p/ F$ |) G5 ]9 K. F2 T3 T) a
+ n5 F: \! e3 W a7 C1 |; pJonassen TEN, Brønd L, Torp M, Græbe M, Nielsen S, Skøtt O, Marcussen N, and Christensen S. Effects of renal denervation on tubular sodium handling in rats with CBL-induced liver cirrhosis. Am J Physiol Renal Physiol 284: F555-F563, 2003.3 {! z! c1 Z1 E2 K, ^
( Q- B" D1 X9 m7 I0 n2 y
9 B, k. U2 L( O! ^) \3 z, d: h8 q! f" L6 C/ }0 ?" f7 @
Jonassen TEN, Christensen S, Kwon TH, Langhoff S, Salling N, and Nielsen S. Renal water handling in rats with decompensated liver cirrhosis. Am J Physiol Renal Physiol 279: F1101-F1109, 2000.6 p& l1 b9 P( I# O3 u
% ~1 n* l ^' }8 E0 B- z, R
1 I: F2 C1 }1 ]
1 F- x) g6 @9 |
Jonassen TEN, Christensen S, Marcussen N, Sørensen N, Flyvbjerg A, Andreasen F, and Petersen JS. Effects of chronic octreotide treatment on renal changes during compensated liver cirrhosis in rats. Hepatology 29: 1387-1395, 1999.
1 }$ q m2 H6 T3 _$ A. H# q" f, O0 q
7 a L5 {+ d- p3 t" r# k4 [9 t* [% {2 l' `) O, K6 }/ K
8 t# A9 Y5 [" {* ^3 R9 ?1 Z0 g
Jonassen TEN, Græbe M, Nielsen S, Promeneur D, Christensen S, and Olsen NV. Lipopolysaccharide-induced acute renal failure in rats: effects of specific phosphodiesterase type 3 and 4 inhibition. J Pharmacol Exp Ther 303: 1-11, 2002.
# d" _/ B3 e7 E+ m; s1 p3 J- W8 F3 \ s, G$ B& X
8 }7 ]5 }; y3 `/ l. j6 W, ?: X+ [4 C& P4 `1 Q; L+ G7 O. {, Q+ q# ?
Jonassen TEN, Marcussen N, Haugan K, Skyum H, Christensen S, Andreasen F, and Petersen JS. Functional and structural changes in the thick ascending limb of Henle's loop in rats with liver cirrhosis. Am J Physiol Regul Integr Comp Physiol 273: R568-R577, 1997.: i; `8 x$ G4 p* l( r
, Z9 W4 m' o& X/ B* B
2 Y$ F7 l3 X; V; P
2 ^/ \' i# \ C+ _# ~- p& N
Jonassen TEN, Nielsen S, Christensen S, and Petersen JS. Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis. Am J Physiol Renal Physiol 275: F216-F225, 1998.
8 y, N- e8 G! r1 m4 X- j& @) _2 W* r' G2 [2 d/ K) V, ~( x$ N+ U+ F: |
6 ]( U# v7 L- S) ?2 N$ d C7 J
}/ F0 R/ w' G: U2 m' eJonassen TEN, Promeneur D, Christensen S, Petersen JS, and Nielsen S. Decreased vasopressin-mediated renal water reabsorption in rats with chronic aldosterone-receptor blockade. Am J Physiol Renal Physiol 278: F246-F256, 2000.0 j$ Y3 I1 p+ k8 o* A. ]
' a7 R3 f. W' B+ O" P
+ ^" @ K% W* W8 O! x n0 n# E5 X. I: G z% }& Y k
Jonassen TEN, Sørensen AM, Petersen JS, Andreasen F, and Christensen S. Increased natriuretic efficiency of furosemide in rats with carbon tetrachloride induced liver cirrhosis. Hepatology 31: 1224-1230, 2000.
/ E9 y& V) i( D) s% Y3 F0 q6 T& C$ r2 t1 `4 g% X4 T5 E4 b$ ~ z
, i9 G8 b: w7 ^9 S; C& Y$ V+ Z& D7 I a0 l
, @; G. q# R1 w5 `$ {# qKjær A, Knigge U, and Warberg J. Dehydration-induced release of vasopressin involves activation of hypothalamic histaminergic neurons. Endocrinology 135: 675-681, 1994.
+ o: y$ ]5 Z/ n& m
9 c) I4 Y; A1 j5 a# D: k1 p
, c; w# s3 l2 g9 b" i+ e1 _2 N: F+ Z' f8 e
Kountouras J, Billing BH, and Scheuer PJ. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 65: 305-311, 1984.( S# B6 Q" T: o
$ z" l& j5 {( V! w% G
6 U/ ]; x, G6 ?8 d& y* X6 [1 Q. X% M3 ]4 d" S' Z( |
Liu H and Maurice DH. Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: differential subcellular localization and regulated expression by cyclic AMP. Br J Pharmacol 125: 1501-1510, 1998.
9 y) s- h* t+ t$ s: q! ]' w) \
2 A# N( O/ L# f/ m6 H2 Y9 Z# P, G! ]# q" u$ b) U4 ?* J7 e
% H& N5 R$ s! P+ |8 W& B% u
Ma T, Hasegawa H, Skach WR, Frigeri A, and Verkman AS. Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel. Am J Physiol Cell Physiol 266: C189-C197, 1994.: D! B8 M/ u+ Y' o9 {. A9 G
( S: c: l, M4 I) w- n
1 O( V3 B% a _$ v& D9 W
% V& K: O+ [* V: |! ?2 U, t9 Q" x
Manning J, Beutler K, Knepper MA, and Vehaskari MV. Upregulation of renal BSC1 and TSC in prenatally programmed hypertension. Am J Physiol Renal Physiol 283: F202-F206, 2002.. [" t& C: z; x4 j# y
2 q, L/ v5 c1 I6 {9 M
* l/ R; u# W" A, |$ ^
2 ~7 ?" y, j/ o$ m Q6 x. k: |Marumo R, Kaizuma S, Nogae S, Kanazawa M, Kimura T, Saito T, Ito S, and Matsubara M. Differential upregulation of rat Na-K-Cl cotransporter, rBSC1, mRNA in the thick ascending limb of Henle in different pathological conditions. Kidney Int 54: 877-888, 1998.
9 p) z4 P* I' B. j S9 W) p
- d, e, Y( E2 ^* J& W1 |' U* F6 J; [$ z, ?3 X; k
/ z+ M2 E' `* x- b8 ]7 F8 G
Miller M. Syndromes of excess antidiuretic hormone release. Crit Care Clin 17: 11-23, 2001.
! T2 t9 }" `- v4 y9 A% g; p8 ^0 B/ S5 X0 K9 G
8 f0 i l/ a% y0 O. C. s
: \& F/ ^ E: B; E, u7 l+ e1 i
Ni XP, Safai M, Gardner DG, and Humphreys MH. Increased cGMP phosphodiesterase activity mediates renal resistance to ANP in rats with bile duct ligation. Kidney Int 59: 1264-1273, 2001.
; U- ]8 v o3 n! ?* k4 o4 v' F) u: E# g; G' G1 X; J
! Q1 i: n& U/ |6 C" W0 \; s
3 j. {0 C, d& [Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, and Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD channels to plasma membrane. Proc Natl Acad Sci USA 92: 1013-1017, 1995.
% L. u* P+ @' T4 A. C
/ q! g+ Z8 ~( C3 [# I! w$ h& L5 I2 X ]* v
; k( r# z! ?6 l( Z* q# P
Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663-11667, 1993.
, Y2 [2 ~$ y0 q' y' G& `: h4 z8 [/ T' O8 _5 d$ W
5 \" v/ t7 K: u, y/ R ?' s8 e: y& M. c% `
Nielsen S, Terris J, Andersen D, Ecelbarger C, Frøkiær J, Jonassen T, Marples D, Knepper MA, and Petersen JS. Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct. Proc Natl Acad Sci USA 94: 5450-5455, 1997.
+ \0 J$ z* h- V6 N' j; \! R9 `- `' ^) E0 R. B) m/ u
6 E- ?5 ]5 j" J3 r: O3 j2 v
* O# a0 ?4 i- X4 w% ]3 l: I
Nogae S, Michimata M, Kanazawa M, Honda S, Ohta M, Imai Y, Ito S, and Matsubara M. Cardiac infarcts increase sodium transporter transcripts (rBSC1) in the thick ascending limb of Henle. Kidney Int 57: 2055-2063, 2000.
8 y# T M: e1 H. z' H* \4 e N; r. u( I+ `: ^6 |# C7 _
, Q* ]9 A$ Y( t2 m# U4 \% P
# H) {7 ?! }' v+ }- uPedersen RS, Bentzen H, Bech JN, Nyvad O, and Pedersen EB. Urinary aquaporin-2 in healthy humans and patients with liver cirrhosis and chronic heart failure during baseline conditions and after acute water load. Kidney Int 63: 1417-1425, 2003.4 s3 K7 ?7 O# ^/ y$ f" b
9 ^/ h+ E! M& [+ f( y4 Z% P
' R0 p5 ]" L5 E. @. I# _( A/ a( q8 t+ e/ O. P" @
Petersen JS, Shalmi M, Lam HR, and Christensen S. Renal response of furosemide in conscious rats: effects of acute instrumentation and peripheral sympathectomy. J Pharmacol Exp Ther 258: 1-7, 1991.
# m) P, \: m( \& Q2 N% e1 g B8 a9 s( V3 L- n4 g, G
' s- J' M0 _ T- t0 l$ L
+ `' |% K4 D& R0 ]& |5 wSabolic I, Katsura T, Verbavatz JM, and Brown D. The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats. J Membr Biol 143: 165-175, 1995.
% I' q7 r" a4 W$ E S
" {% n: c4 @% E0 b( w
" E l9 |; k; q3 i# E- e; p7 @8 D
! `5 B$ W! C0 ^7 {Schrier RW, Gurevich AK, and Cadnapaphornchai MA. Pathogenesis and management of sodium and water retention in cardiac failure and cirrhosis. Semin Nephrol 21: 157-172, 2001., M) v- ?% a( e# d2 P* W! Z( w p
% [7 q6 M8 Z9 Y# z* L+ e0 }' Z" T, ]
% p+ R6 K4 l0 N8 c. ^* v2 N
Staahltoft D, Nielsen S, Janjua NR, Christensen S, Marcussen N, Skøtt O, and Jonassen TEN. Chronic losartan treatment normalizes renal water handling in rats with congestive heart failure. Am J Physiol Renal Physiol 282: F307-F315, 2002.
/ M( V7 e, }' e9 T
; O, C" L8 S' [' e4 ^3 z" k$ l( N9 @" n, v
* d: w( k; }5 R
Tian Y, Sandberg K, Murase T, Baker EA, Speth RC, and Verbalis JG. Vasopressin V 2 receptor binding is downregulated during renal escape from vasopressin-induced antidiuresis. Endocrinology 141: 307-314, 2000.: D9 z# W' m4 ^" _+ A
7 w- e: y9 F# |" u5 f8 d% a! A
; w/ m+ ?- O9 j+ s9 ]2 {
4 a+ N: n) i" ?; wXu DI, Martin PY, Ohara M, St. John J, Pattison T, Meng X, Morris K, Kim JK, and Schrier RW. Upregulation of aquaporin-2 water channel expression in chronic heart failure in rat. J Clin Invest 99: 1500-1505, 1997.! e% G7 b/ P* r4 z. v& c
2 u p- ~3 l* `$ G( G/ G, c/ A/ e$ ~
6 o/ o3 I$ P6 _1 e4 N" ]' c
Yasui M, Zelenin SM, Celsi G, and Aperia A. Adenylate cyclase-coupled vasopressin receptor activates AQP2 promoter via a dual effect on CRE and AP1 elements. Am J Physiol Renal Physiol 272: F443-F450, 1997. K5 E' V) t4 a$ K: n$ Z
5 B( w. Z8 [+ _8 Q1 n$ m) U+ G# q# v, f, n2 \* i, C* g
# u- N; ?1 q2 q& M6 e! _4 SYoshida I, Takeda S, Homma S, Kusano E, and Asano Y. Localization of low- K m cAMP phosphodiesterase in rat nephron segments. Kidney Blood Press Res 20: 307-311, 1997. |
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发表于 2009-4-22 08:09
