1Department of Pharmacology, University of Copenhagen, DK-2200 Copenhagen N; and 2The Water and Salt Research Center, Institute of Anatomy (Building 233), University of Aarhus, DK-8000 Aarhus C, Denmark
Submitted 7 August 2003 ; accepted in final form 26 May 2004
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ABSTRACT |
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aquaporin-2; phosphodiesterase; common bile duct ligation; kidney; cAMP
Liver 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.
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.
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 V2 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.
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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METHODS |
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Female Wistar rats (230250 g) from Charles River (Sulzfeld, Germany) were used for the experiments. The animals were housed in a temperature (2224°C)- and moisture (4070%)-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.
Animal Preparation
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.
Series 1
Measurement 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).
Isolation of CDs.
After blood sample collection, the rats were anesthetized by halothane in N2O-O2 (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 O2-aerated microdissection solution (135 mM NaCl, 1.0 mM Na2HPO4, 2 H2O, 5.0 mM KCl, 1.2 mM MgSO4, 2.0 mM CaCl2, 1.2 mM NaSO4, 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 O2 aeration. The incubation time was 2425 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.
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 MgSO4, 7 H2O, 10 mM sodium acetate, 10 mM D+-glucose monohydrat, 20 mM Tris base, 2 mM Na2HPO4, 2 H2O, 0.8 mM CaCl2, 2 H2O was added to the samples (the osmolarity of the buffer should be 280 mosM). For basal CD cAMP accumulation, 1012 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: 106 M (3- to 4-mm CD), 108 M (5- to 6-mm CD), 109 M (8- to 9-mm CD), and 1010 M (9- to 10-mm CD).
Finally, 1.5- to 2-mm CD was incubated in a medium containing AVP 106 and 5·104 M of the nonspecific PDE inhibitor IBMX.
To ensure correct pH in the incubation media, the solutions were aerated with 95% O2-5% CO2 and the pH was measured just before using the media.
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.
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.
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.
The 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).
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).
Series 2
Immunohistochemisty. Four weeks after CBL or sham operation, another series of animals (n = 5 in both groups) was anesthetized by isoflurane in N2O/O2 (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 8x8-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 4x8-pixel part of the 8x8-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.
Series 3
Western blotting of PDE. Four weeks after CBL or sham operation, rats were anesthetized by isoflurane in N2O/O2 (2:1) and both kidneys were rapidly removed, frozen in liquid nitrogen, and stored at 80°C until processing for membrane fractionation.
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).
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.
For 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.
We obtained specific binding for the following PDE isoforms: PDE4B4 (66 kDa), PDE4D1 and 2 (68 kDa), and PDE4D4 (119 kDa).
Series 4
Measurement 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).
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.
Statistics
Data 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 |
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Figure 1B 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. 1C) 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 < 0.05). In addition, Fig. 1D shows a Western blot of pAQP2. As previously shown (7), the affinity-purified anti-pAQP2 protein antibody (AN244) also recognizes a 29- and a 35- to 50-kDa band, corresponding to the nonglycosylated and glycosylated pAQP2 protein, respectively. Densitometry (Fig. 1E) showed that the amount of pAQP2 was significantly decreased in the cirrhotic rats (0.50 ± 0.06 of the sham rats; P < 0.05).
Immunohistochemical Visualization of AQP2 in the CDs
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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To address the question of whether the uncoupling of V2 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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It is well described that AVP through activation of V2 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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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.
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 < 0.05; Fig. 6A]. In addition, comparing sham thirst and CBL thirst, we found the same level of PDE4B4 downregulation [sham thirst: 1 ± 0.05 (n = 7) vs. CBL thirst: 0.51 ± 0.05 (n = 7); P < 0.05; Fig. 6B]. Finally, thirsting induced comparable decreases within the PDE4B4 expression of the CBL and sham groups [CBL: 1 ± 0.03 (n = 5) vs. CBL thirst: 0.77 ± 0.04 (n = 7); P < 0.05; sham: 1 ± 0.04 (n = 6) vs. sham thirst: 0.73 ± 0.04 (n = 7); P < 0.05; Fig. 6, C and D]. This suggests that decreased PDE4B4 expression might be involved in the increased V2 receptor signaling present during thirsting.
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DISCUSSION |
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If 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.
A 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 V2 receptor agonist (dDAVP) treatment. In this model, AVP escape seems to be associated with downregulation of V2 receptor binding and decreased cellular cAMP accumulation along with decreased AQP2 protein and mRNA levels (8, 39).
The 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 V2 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.
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 CCl4 treatment (4, 12). It could be asked whether the discrepancies between these findings within the CCl4 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 CCl4 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 CCl4 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 CCl4-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.
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.
The 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.
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.
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.
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.
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.
Finally, 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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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