Increased Apical Targeting of Renal Epithelial Sodium Channel Subunits and Decreased Expression of Type 2 11-Hydroxysteroid Dehydrogenase in Rats with CCl4-Induced Decompensated Liver Cirrhosis
Soo Wan Kim*,,
Uffe K. Schou*,
Christian D. Peters*,
Sophie de Seigneux*,
Tae-Hwan Kwon*,,
Mark A. Knepper,
Thomas E.N. Jonassen||,
Jørgen Frøki* and
Søren Nielsen*
* The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark; Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea; Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and || Department of Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
Address correspondence to: Dr. Søren Nielsen, The Water and Salt Research Center, Building 233/234, University of Aarhus, Aarhus, Denmark DK-8000. Phone: +45-8942-3046; Fax: +45-8619-8664; sn{at}ana.au.dk
Received for publication August 31, 2004.
Accepted for publication August 9, 2005.
It was hypothesized that dysregulation of renal epithelial sodiumchannel (ENaC) subunits and/or 11-hydroxysteroid dehydrogenase(11HSD2) may play a role in the increased sodium retention inliver cirrhosis (LC). Experimental LC was induced in rats byCCl4 (1 ml/kg, intraperitoneally, twice a week) for 12 wk (protocol1) or for 11 wk (protocol 2). In both protocols, one group ofrats with cirrhosis showed significantly decreased urinary sodiumexcretion and urinary Na/K ratio (group A), whereas a secondgroup exhibited normal urinary sodium excretion (group B) comparedwith controls, even though extensive ascites was seen in bothgroups of rats with cirrhosis. In group A, protein abundanceof -ENaC was unchanged, whereas -ENaC abundance was decreasedin the cortex/outer stripe of outer medulla compared with controls.The -ENaC underwent a complex change associated with increasedabundance of the 70-kD band with a concomitant decrease in themain 85-kD band, corresponding to an aldosterone effect. Incontrast, no changes in the abundance of ENaC subunit were observedin group B. Immunoperoxidase microscopy revealed an increasedapical targeting of -, -, and -ENaC subunits in distal convolutedtubule (DCT2), connecting tubule (CNT), and cortical and medullarycollecting duct segments in group A but not in group B. Immunolabelingintensity of 11HSD2 in the DCT2, CNT, and cortical collectingduct was significantly reduced in group A but not in group B,and this was confirmed by immunoblotting. In conclusion, increasedapical targeting of ENaC subunits combined with diminished abundanceof 11HSD2 in the DCT2, CNT, and cortical collecting duct islikely to play a role in the sodium retaining stage of livercirrhosis.
Renal sodium and water retention has been shown to be responsiblefor the development of ascites not only in patients with livercirrhosis but also in experimental rats with cirrhosis. Atrialnatriuretic peptide resistance (1), arginine vasopressin (AVP)(2), renin-angiotensin-aldosterone (3), and sympathetic nerveoveractivity (4) have been shown to modulate renal tubular functionsand have been considered to be involved mainly in sodium andwater retention in liver cirrhosis. In kidneys of rats withcirrhosis, increased reabsorption of sodium and water in thedistal nephron and collecting duct has been suggested to beone of the most important renal tubular dysfunctions involvedin the pathogenesis of ascites (5–7). However, the underlyingmolecular and cellular mechanisms for the sodium retention arestill incompletely understood.
In particular, the role of aldosterone in sodium retention andascites formation in liver cirrhosis is still unclear. Previousstudies demonstrated that plasma aldosterone levels were usuallyelevated in liver cirrhosis with ascites (8–10) and thatspironolactone, a mineralocorticoid receptor antagonist, increasedsodium excretion in these patients (11). This suggests thathyperaldosteronism is of major importance in the pathogenesisof sodium retention. The epithelial sodium channel (ENaC) isthe major sodium transport pathway in the collecting duct (12),and both protein abundance and apical plasma membrane targetingof the ENaC subunits are regulated by hormonal stimulation,e.g., aldosterone (13) and vasopressin (14). We therefore speculatedthat altered protein abundance and/or apical membrane targetingof ENaC subunits (, , and ) may account for the increased sodiumreabsorption in the collecting duct and sodium retention inliver cirrhosis. However, other investigators have demonstratedthat plasma aldosterone levels were normal in patients who hadcirrhosis and presented ascites and that sodium retention persisteddespite the maintenance of plasma aldosterone levels withinnormal limits (9,15). Thus, it would also be possible that theincreased sodium reabsorption in the distal nephron and collectingduct could be mediated by other regulatory mechanisms. The intracellularaccess of glucocorticoids to mineralocorticoid receptors (MR)is modulated by the type 2 isoform of 11-hydroxysteroid dehydrogenase(11HSD2). 11HSD2 confers mineralocorticoid specificity in aldosterone-sensitiveepithelia in the kidney by metabolizing glucocorticoids, thusprotecting MR from inappropriate activation by glucocorticoid(16,17). Such protection is critical because the glucocorticoidcortisol circulates in a 100-fold molar excess to aldosteronein vivo and binds to MR with an affinity similar to aldosterone(18,19). The importance of the enzyme is illustrated by theclinical consequences of mutations (20,21) in the 11HSD2 geneand by the use of inhibitors (22) of 11HSD2, both of which leadto the syndrome of apparent mineralocorticoid excess, a rareform of hypertension with low plasma aldosterone and sodiumretention. Thus, we speculated that an increased access of cortisolto the MR in a condition that demonstrated decreased activity/expressionof 11HSD2 could account, at least in part, for the enhancedaldosterone effect in decompensated cirrhosis, although plasmaaldosterone levels are not increased.
The purposes of this study, therefore, were (1) to examine whetherthere are changes in the protein abundance and/or apical targetingof ENaC subunits in kidneys of rats with CCl4-induced livercirrhosis, (2) to examine whether there are changes in the proteinabundance of 11HSD2, (3) to examine whether there are changesin the protein abundance of other renal sodium transportersalong the renal tubules (type 3 Na/H exchanger [NHE3] and Na-K-2Clco-transporter [NKCC2]), and (4) to examine whether these changesare associated with changes in the urinary sodium excretion.
Experimental Protocols Protocol 1 (12 Wk of CCl4 Injection).
Experiments were performed using male Munich-Wistar rats (200to 250 g; Møllegard Breeding Centre, Ll. Skensved, Denmark).The animal protocols were approved by the boards of the Instituteof Anatomy and Institute of Clinical Medicine, University ofAarhus, according to the licenses for use of experimental animalsissued by the Danish Ministry of Justice. Liver cirrhosis (n= 10) was induced by intraperitoneal injections of a solutionof CCl4 in groundnut oil (1:1), 1 ml/kg body wt twice a weekthroughout the experimental period. Control rats (n = 7) receivedintraperitoneal injections of groundnut oil 0.5 ml/kg body wt.For accelerating the generation of cirrhosis, all rats (bothcontrol and CCl4-treated groups) received phenobarbital in thedrinking water (350 mg/L) throughout the whole experimentalperiod (23). They were maintained in individual cages on a standardrodent diet (Altromin #1324; Altromin, Lage, Germany) and allowedfree access to drinking water at all times. CCl4-treated andcontrol rats were pair fed. In the control group, rats wereoffered the amount of food corresponding to the mean intakeof food that the CCl4-treated rats consumed during the previousday.
During the last 3 d, the rats were subsequently maintained inthe metabolic cages to allow urine collections for the measurementsof Na+, K+, creatinine, and osmolality. The rats were killedfor immunoblotting and immunohistochemical studies 12 wk afterCCl4 treatment. Rats were anesthetized with halothane (HalocarbonLaboratories, River Edge, NJ), and a large laparotomy was made.Peritoneal fluid volume was quantified in each rat by absorbingthe ascites fluid into preweighed dry papers and reweighingthe papers. The difference in weight represented the weightof the collected peritoneal fluid. Blood was collected fromthe inferior vena cava and analyzed for bilirubin, alanine aminotransaminase(ALT), Na+, K+, creatinine, osmolality, and plasma aldosteroneconcentration using methods described previously in detail (24).The right kidney was rapidly removed, dissected into three zones(cortex and outer stripe of outer medulla [OSOM], inner stripeof outer medulla [ISOM], and inner medulla) and processed forimmunoblotting as described below. The left kidney was fixedby retrograde perfusion as described below.
Protocol 2 (11 Wk of CCl4 Injection).
To confirm the changes of subcellular redistribution of ENaCsubunits in the sodium-retaining stage of liver cirrhosis observedin protocol 1, another set of CCl4-treated rats (n = 11) andcontrol rats (n = 7) was made. This protocol was identical toprotocol 1, except that control and CCl4-treated rats were monitoredfor 11 wk.
Subgroups in CCl4-Treated Rats
CCl4-treated rats displayed various renal responses in the renalsodium retention/excretion. Some rats showed markedly decreasedurinary sodium excretion (sodium retaining stage, group A),whereas the others did not exhibit differences in urinary sodiumexcretion (maintenance stage, group B) compared with controls,even though all CCl4-treated rats in both groups had a significantamount of ascites. Thus, we subdivided rats with liver cirrhosisinto two groups (group A and group B), according to the urinarysodium excretion measured at the last day of the experiment(Figure 1).
Figure 1. Urinary sodium excretion (A) and urinary Na/K ratio (B) from control and CCl4-treated rats with cirrhotic (Cirrhosis) in protocol 1. (A) Urinary sodium excretion was similar in control and rats with CCl4-induced cirrhosis. Four (; group A) of the rats with CCl4-induced cirrhosis showed markedly decreased urinary sodium excretion, whereas the other six rats (; group B) remained similar compared with controls. (B) The urinary Na/K ratio was decreased in rats with cirrhosis, indicating increased aldosterone effectiveness in the distal nephron. *P < 0.05 versus control.
Semiquantitative Immunoblotting
The dissected renal cortex/OSOM, ISOM, and inner medulla werehomogenized (Ultra-Turrax T8 homogenizer; IKA Labortechnik,Staufen, Germany) in ice-cold isolation solution that contained0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin,and 1 mM PMSF (pH 7.2). The homogenates were centrifuged at4000 x g for 15 min at 4°C to remove whole cells, nucleiand mitochondria, and the supernatant was pipetted off and kepton ice. The total protein concentration was measured (PierceBCA protein assay reagent kit; Pierce, Rockford, IL). All sampleswere solubilized at 65°C for 15 min in SDS-containing samplebuffer. For confirming equal loading of protein, an initialgel was stained with Coomassie Blue. SDS-PAGE was performedon 9 or 12% polyacrylamide gels. The proteins were transferredby gel electrophoresis (BioRad Mini Protean II) onto nitrocellulosemembranes (Hybond ECL RPN3032D; Amersham Pharmacia Biotech,Little Chalfont, UK). The blots were subsequently blocked with5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl,and 0.1% Tween 20 [pH 7.5]) for 1 h and incubated overnightat 4°C with primary antibodies followed by incubation withanti-rabbit (P448; DAKO, Glostrup, Denmark), anti-mouse (P447;DAKO), or anti-sheep (1713–035-147; Jackson Laboratories,Bar Harbor, ME) horseradish peroxidase–conjugated secondaryantibodies. The labeling was visualized by an enhanced chemiluminescencesystem (ECL or ECL+plus) and exposure to photographic film (HyperfilmECL, Amersham). ECL films were scanned using an AGFA scanner(ARCUS II).
Immunohistochemistry
For perfusion fixation, a perfusion needle was inserted in theabdominal aorta of rats and the vena cava was cut to establishan outlet. Blood was flushed from the kidneys with cold PBS(pH 7.4) for 15 s before switching to cold 3% paraformaldehydein 0.1 M cacodylate buffer (pH 7.4) for 3 min. The kidney andliver were removed and sectioned into 2- to 3-mm transversesections and immersion fixed for an additional 1 h, followedby 3 x 10-min washes with 0.1 M cacodylate buffer of pH 7.4.The tissue was dehydrated in graded ethanol and left overnightin xylene. After embedding in paraffin, 2-µm sectionsof the tissue were cut on a rotary microtome (Leica MicrosystemsA/S, Herlev, Denmark).
Immunolabeling was performed on sections from paraffin-embeddedpreparation (2 µm thickness) using methods described previouslyin detail (24). For the liver histology, sections were stainedwith hematoxylin-eosin. Light microscopy was carried out withLeica DMRE (Leica Microsystems A/S).
Primary Antibodies
We used previously characterized polyclonal antibodies. Affinity-purifiedpolyclonal antibodies against the renal sodium transporters/channels(24) (NHE3, NKCC2, thiazide-sensitive Na-Cl co-transporter [NCC],the ENaC subunits), aquaporin 2 (AQP2) and phosphorylated-AQP2(p-AQP2) (25) were used. A sheep polyclonal antibody to 11HSD2(Chemicon, Temecula, CA) was commercially obtained. A mousemAb against the Na,K-ATPase 1-subunit was provided by Dr. D.M.Fambrough (Johns Hopkins University Medical School, Baltimore,MD).
Statistical Analyses
Values are presented as means ± SE. Comparisons betweentwo groups were made by the unpaired t test. Multiple comparisonsamong the groups were made by one-way ANOVA and post hoc TukeyHSD test. Multiple-comparisons tests were applied only whena significant difference was determined in the ANOVA (P <0.05). P < 0.05 were considered significant.
Changes of Urinary Sodium Excretion in CCl4-Treated Rats (12-Wk Experiment, Protocol 1) Table 1 shows the changes in liver and renal function in the12-wk experiment (protocol 1). All of the CCl4-treated ratshad large amount of ascites. Light microscopy of liver histologyrevealed well-established liver cirrhosis in all CCl4-treatedrats, in which the combination of nodular regeneration of livercell plates surrounded by thick connective tissue septa withproliferating bile ducts define a picture of micronodular cirrhosis(data not shown) (26). Consistent with this, plasma concentrationsof ALT and bilirubin were significantly increased in CCl4-treatedrats. Plasma creatinine levels were decreased, whereas renalcreatinine clearance was not changed in CCl4-treated rats. The24-h urinary sodium excretion showed a decreased tendency inrats with cirrhosis. However, it was not statistically significantbecause the rats with cirrhosis showed wide variations in urinarysodium excretion (Figure 1A). Importantly, fractional excretionof sodium was decreased, and sodium balance was positive inCCl4-treated rats. Urinary Na/K ratio was also decreased inCCl4-treated rats. Moreover, the urinary osmolality (1934.0± 128 versus 1549.7 ± 71 mOsm/kgH2O in controls;P < 0.05) and urine/plasma osmolality ratio were increased(6.6 ± 0.4 versus 5.1 ± 0.2 in controls; P <0.01), indicating increased urinary concentration in CCl4-treatedrats.
Table 1. Changes in liver and renal function (12-wk experiment, protocol 1)a
Altered Protein Expression of ENaC Subunits in CCl4-Treated Rats (12-Wk Experiment, Protocol 1)
In protocol 1, the protein abundance of -ENaC was unchangedin cortex/OSOM, ISOM, and inner medulla in CCl4-treated ratscompared with controls (Figure 2). The abundance of -ENaC wasdecreased in the cortex/OSOM, ISOM, and inner medulla (Figure 2).The -ENaC underwent a complex change associated with theappearance of a 70-kD band with a concomitant decrease in themain 85-kD band. These changes were prominent, especially inthe CCl4-treated rats associated with markedly decreased urinarysodium excretion (group A; Figure 2, arrows). The summary ofanalyses of normalized band densities is shown in Table 2.
Figure 2. Semiquantitative immunoblots of kidney protein prepared from cortex/outer stripe of outer medulla (OSOM), inner stripe of outer medulla (ISOM), and inner medulla from control rats and CCl4-treated rats with cirrhosis in protocol 1. The subunit of the epithelial sodium channel (-ENaC) band at 85 kD was maintained, and the -ENaC band at 85 kD was decreased in the cortex/OSOM, ISOM, and inner medulla. The -ENaC underwent a complex change. These perturbations were associated with the appearance of a 70-kD band with a concomitant decrease in the main 85-kD band. These changes were prominent, especially in the CCl4-treated rats associated with markedly decreased urinary sodium excretion (arrows indicate the group 1 rats). *P < 0.05 versus control.
In protocol 1 of 12 wk of CCl4 treatment, four rats (group A)out of all of the CCl4-treated rats showed markedly decreasedurinary sodium excretion and urinary Na/K ratio, whereas theother six rats (group B) exhibited unchanged urinary sodiumexcretion and urinary Na/K ratio compared with controls (Figure 1,Table 3). Plasma aldosterone levels were markedly increasedin group A, whereas the levels were decreased in group B comparedwith controls (Table 3). Thus, we subdivided CCl4-treated ratsinto group A and group B according to urinary sodium excretion.To investigate whether the changes of protein abundance of ENaCsubunits are related with the sodium retention, we performedadditional immunoblot analysis in the cortex/OSOM (Figure 3).In group A, protein abundance of -ENaC was unchanged, whereas-ENaC abundance was decreased in the cortex/OSOM compared withcontrols. The -ENaC underwent a complex change associated withthe increased abundance of the 70-kD band with a concomitantdecrease in the main 85-kD band (Figure 3). In contrast, therewere no significant changes of abundance of ENaC subunits ingroup B (Figure 3).
Figure 3. Semiquantitative immunoblots of kidney protein prepared from cortex/OSOM of control and CCl4-treated rats with cirrhosis subdivided into group A or group B liver cirrhosis in protocol 1. In group A, protein abundance of -ENaC was unchanged, whereas -ENaC abundance was decreased compared with controls. The -ENaC underwent a complex change associated with the increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band. In contrast, there were no significant changes of ENaC subunit expression in group 2. *P < 0.05 versus control; #P < 0.05 versus group 1.
Increased Apical Targeting of ENaC Subunits in Liver Cirrhosis (Group A, Protocol 1)
To investigate whether the trafficking of ENaC subunits is alteredin CCl4-treated rats, we carried out immunoperoxidase microscopyof ENaC subunits. According to the light microscopic findings,we can differentiate the tubular segmental distributions. Thedistal convoluted tubule (DCT) cells contain numerous mitochondriain the cytoplasm and the nuclei that occupy middle to apicalposition. The connecting tubule (CNT) cells are intermediatein ultrastructure between the DCT and the collecting duct principalcell. They are taller than principal cells of collecting duct.DCT and CNT are located in the cortical labyrinth. Collectingduct principal cells in the cortex are cuboidal in rats. Theyhave a simple cell shape with light-staining cytoplasm, withfairly straight lateral cell borders, and nucleus is situatedin the upper half of the cell in the cortex. The cortical collectingduct (CCD) can be subdivided further into two parts: InitialCCD in the cortical labyrinth and the collecting duct locatedin the medullary ray. We have examined the CCD in the medullaryray only because initial CCD was not easy to distinguish fromCNT in the cortical labyrinth. To confirm the tubule segments,double labeling with -ENaC and calbindin-D28k (a marker forDCT and CNT segments) was carried out and analyzed by laserscanning confocal microscopy, as described previously in detail(24).
In control rats, immunoperoxidase staining for the -ENaC subunitsshowed diffuse cytoplasmic labeling throughout the DCT, CNT,and collecting duct principal cells (Figure 4, A, D, G, andJ). In contrast, rats in group A of liver cirrhosis revealed-ENaC labeling predominantly localized to the apical plasmamembrane domains with weaker cytoplasmic labeling. This wasevident in all of the cross-sectioned tubules of DCT2 (Figure 4B),CNT (Figure 4E), CCD (Figure 4H), and outer medullary collectingduct (OMCD; Figure 4K). On the contrary, rats in group B ofliver cirrhosis had a similar immunolabeling pattern of -ENaC(Figure 4, C, F, I, and L) to the control rats (Figure 4, A,D, G, and J).
Figure 4. Immunoperoxidase microscopy of -ENaC in the second half of distal convoluted tubule (DCT2), connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in protocol 1. Immunoperoxidase labeling of -ENaC is dispersed in the cytoplasm of principal cells of the DCT2 (A), CNT (D), CCD (G), and OMCD (J) in control rats. In contrast, -ENaC labeling was seen predominantly localized to the apical plasma membrane domains, and only marginal cytoplasmic labeling was observed in DCT2 (B), CNT (E), CCD (H), and OMCD (K) in group A. However, the immunolabeling pattern of -ENaC was similar in group B liver cirrhosis compared with controls.
Immunohistochemical analysis also revealed changes in the subcellularredistribution of -ENaC in kidneys from CCl4-treated rats similarto the changes of -ENaC. There was a marked increase in apical-ENaC immunolabeling in DCT2 (Figure 5B), CNT (Figure 5E), CCD(Figure 5H), and OMCD (Figure 5K) in kidneys from group A. Incontrast, the immunolabeling pattern of -ENaC in group B (Figure 5,C, F, I, and L) was unchanged (Figure 5, A, D, G, and J).Consistently, immunoperoxidase microscopy revealed an increasedapical immunolabeling of -ENaC in DCT2 (Figure 6B), CNT (Figure 6E),CCD (Figure 6H), and OMCD (Figure 6K) in group A but notin group B (Figure 6, C, F, I, and L).
Figure 5. Immunoperoxidase microscopy of -ENaC in DCT2, CNT, CCD, and OMCD in protocol 1. Immunoperoxidase labeling of -ENaC is associated mainly with the entire cytoplasm of principal cells of the DCT2 (A), CNT (D), CCD (G), and OMCD (J) in control rats. In contrast, -ENaC labeling was markedly redistributed to the apical plasma membrane domains in DCT2 (B), CNT (E), CCD (H), and OMCD (K) in group A cirrhosis. However, the immunolabeling pattern of -ENaC was similar in group B liver cirrhosis compared with controls.
Figure 6. Immunoperoxidase microscopy of -ENaC in DCT2, CNT, CCD, and OMCD in protocol 1. Immunoperoxidase labeling of -ENaC is restricted to a narrow zone in the apical part, including the plasma membrane domains of the principal cells of the DCT2 (A), CNT (D), CCD (G), and OMCD (J) in control rats. In contrast, group A rats with cirrhosis showed markedly increased apical immunolabeling of -ENaC in DCT2 (B), CNT (E), CCD (H), and OMCD (K). However, the immunolabeling pattern of -ENaC was similar in group B liver cirrhosis compared with controls.
Changes of Urinary Sodium Excretion in CCl4-Treated Rats (11-Wk Experiment, Protocol 2) Table 4 shows the changes in liver and renal function in protocol2 (11-wk experiment). All of the CCl4-treated rats had significantamount of ascites. Plasma concentrations of ALT and bilirubinwere significantly increased in CCl4-treated rats. Plasma creatinineand renal creatinine clearance were not changed in CCl4-treatedrats. The 24-h urinary sodium excretion and fractional excretionof sodium were decreased, and accordingly sodium balance waspositive in CCl4-treated rats (Table 4).
Table 4. Changes in liver and renal function (11-wk experiment, protocol 2)a
In protocol 2 of 11 wk of CCl4 treatment, we subdivided CCl4-treatedrats into two groups (group A and group B) according to theurinary sodium excretion, similar to protocol 1. Four rats (groupA) out of all of the CCl4-treated rats showed markedly decreasedurinary sodium excretion and urinary Na/K ratio compared withcontrols, whereas the other seven rats (group B) had unchangedurinary sodium excretion and Na/K ratio (Table 5). In contrastto protocol 1, plasma aldosterone levels were not changed ingroup A. The levels were decreased in group B compared withcontrols (Table 5).
Table 5. Subgroup analysis of urinary sodium excretion and plasma aldosterone levels (11-wk experiment, protocol 2)a
Altered Regulation of ENaC Abundance and Trafficking (11-Wk Experiment, Protocol 2)
In protocol 2, the major changes of abundance and subcellulardistribution of ENaC subunits were virtually identical to thoseof protocol 1. The protein abundance of -ENaC and -ENaC wasunchanged. However, the -ENaC underwent a complex change associatedwith the increased abundance of the 70-kD band with a concomitantdecrease in the main 85-kD band in group A but not in groupB (Figure 7A). There was a prominent increase in apical traffickingof -ENaC and -ENaC in DCT2, CNT, CCD, and OMCD in group A butnot in group B. Representative immunoperoxidase labeling of-ENaC in the CCD showed an increased apical labeling in groupA, whereas no change of subcellular distribution of -ENaC wasobserved in group B compared with controls (Figure 7B).
Figure 7. Semiquantitative immunoblots (A) and immunoperoxidase microscopy (B) of ENaC subunits prepared from cortex/OSOM of control and CCl4-treated rats with cirrhosis (11 wk) in protocol 2. (A) In group A, protein abundance of -ENaC and -ENaC was unchanged compared with controls, whereas -ENaC underwent a complex change associated with the increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band. There were no significant changes of ENaC subunit expression in group B. (B) Immunoperoxidase labeling of -ENaC is dispersed in the cytoplasm of principal cells of the CCD in control rats. The labeling is markedly increased in the apical plasma membrane domains in group A, whereas the subcellular distribution of -ENaC was similar in group B liver cirrhosis compared with controls. *P < 0.05 versus control; #P < 0.05 versus group A.
Decreased Protein Abundance of 11HSD2 in CCl4-Treated Rats
Semiquantitative immunoblotting was carried out to investigatewhether the abundance of 11HSD2 was altered in rats with CCl4-inducedliver cirrhosis. In protocol 1, the protein abundance of 11HSD2was decreased in the cortex/OSOM (51 ± 8 versus 100 ±16% in controls; P < 0.05; Figure 8A) but not changed inISOM (93 ± 6 versus 100 ± 5% in controls; NS)and inner medulla (138 ± 41 versus 100 ± 23% incontrols; NS).
Figure 8. Semiquantitative immunoblotting of the type 2 isoform of 11-hydroxysteroid dehydrogenase (11HSD2) from cortex/OSOM in protocol 1 (A and B) and protocol 2 (C). (A) Protein abundance of 11HSD2 in the cortex/OSOM was significantly reduced in CCl4-treated rats compared with control rats (12 wk). (B) Compared with controls, the abundance of 11HSD2 was markedly decreased in cortex/OSOM in group A but not in group B compared with controls (12 wk). (C) Similar changes were observed in 11-wk CCl4-treated rats with cirrhosis. The 11HSD2 expression was markedly decreased in cortex/OSOM in group A but not in group B. *P < 0.05 versus control.
To investigate whether there were changes in the protein abundanceand/or immunolabeling of 11HSD2 among group A and group B ofliver cirrhosis and controls, we performed additional immunoblottingand immunocytochemical analysis. Compared with controls, theabundance of 11HSD2 was markedly decreased in the cortex/OSOMin group A from both protocols (Figure 8, B [protocol 1] andC [protocol 2]) but not in group B. Immunolabeling of 11HSD2was associated with DCT2, CNT, and collecting duct principalcells. Immunoperoxidase microscopy revealed cytoplasmic stainingof 11HSD2 that was observed mainly at the perinuclear region(Figure 9). However, immunogold electron microscopy for 11HSD2in the kidney of control rats revealed that immunogold labelingwas diffusely distributed to whole cytoplasm and was not concentratedto the perinuclear region (data not shown). Importantly, ratsin group A from protocol 1 demonstrated decreased immunolabelingintensity of 11HSD2 in DCT2 (Figure 9B), CNT (Figure 9E), andCCD (Figure 9H). In contrast, rats in group B from protocol1 did not reveal any significant changes of the immunolabelingof 11HSD2 in the DCT2 (Figure 9C), CNT (Figure 9F), and CCD(Figure 9I), compared with control rats (Figure 9, A, D, andG). In contrast, immunolabeling intensity of 11HSD2 in OMCDdid not show any significant changes among the three groups(Figure 9, J, K, and L).
Figure 9. Immunoperoxidase microscopy of 11HSD2 in DCT2, CNT, CCD, and OMCD in protocol 1. Immunoperoxidase labeling of 11HSD2 was dispersed in the cytoplasm of principal cells of the DCT2 (A), CNT (D), CCD (G), and OMCD (J) in control rats. In group A rats with cirrhosis, the immunolabeling intensity of 11HSD2 was markedly decreased in DCT2 (B), CNT (E), and CCD (H). However, group B rats with liver cirrhosis did not show any significant changes of the immunolabeling intensity in the DCT2 (A and C), CNT (D and F), and CCD (G and I) compared with control rats. Immunolabeling intensity of 11HSD2 in OMCD did not show any significant changes among three groups (J, K, and L).
Altered Protein Abundance of Major Renal Sodium Transporters in CCl4-Treated Rats (12-Wk Experiment, Protocol 1)
Protein abundance of the 1-isoform of Na,K-ATPase was unchangedin the cortex/OSOM, ISOM, and inner medulla (Table 6). The NHE3abundance was decreased in the cortex/OSOM but was increasedin the ISOM (Figure 10, Table 6). Immunocytochemistry demonstratedthat the labeling intensity of NHE3 in the proximal tubule ofCCl4-treated kidney was reduced but was increased in medullarythick ascending limb (mTAL) compared with control rats (datanot shown). The protein abundance of NKCC2 was increased inISOM (Figure 10) in CCl4-treated rats but was not altered inthe cortex/OSOM (Table 6). In contrast to the significant changesin renal protein abundance of NHE3 and NKCC2, the NCC abundancewas unchanged in the cortex/OSOM (Table 6).
Figure 10. Semiquantitative immunoblotting of kidney protein prepared from ISOM of control and CCl4-treated rats with cirrhosis in protocol 1. (A) Immunoblot was reacted with anti–type 3 Na/H exchanger (NHE3) antibodies. (B) Densitometric analyses revealed that NHE3 expression in ISOM was increased in CCl4-treated rats compared with control rats. (C) Immunoblots were reacted with anti–Na-K-2Cl co-transporter (NKCC2) antibodies. (D) Densitometric analyses revealed that NKCC2 expression in ISOM was increased in CCl4-treated rats compared with control rats. *P < 0.05 versus control.
Increased Protein Abundance and Apical Plasma Membrane Targeting of AQP2 and p-AQP2 in CCl4-Treated Rats (12-Wk Experiment, Protocol 1)
Semiquantitative immunoblotting revealed an increase in AQP2abundance in the inner medulla (221 ± 21 versus 100 ±14% in controls; P < 0.05; Figure 11, A and B), whereas therewere no changes in the cortex/OSOM (115 ± 10 versus 100± 15% in controls; NS) and ISOM (114 ± 6 versus100 ± 9%; NS). Moreover, semiquantitative immunoblottingwith antibodies that selectively recognize AQP2 (p-AQP2), whichis phosphorylated in the protein kinase A phosphorylation consensussite (Ser256) (25), demonstrated that the abundance of p-AQP2was also increased in the inner medulla (198 ± 28 versus100 ± 15%; P < 0.05) but was not changed in the cortex/OSOM(100 ± 7 versus 100 ± 9%; NS) and ISOM (98 ±7 versus 100 ± 15%; NS).
Figure 11. Semiquantitative immunoblotting and immunoperoxidase microscopy of kidney protein prepared from inner medulla of control and CCl4-treated rats with cirrhosis in protocol 1. (A) Immunoblot was reacted with anti–aquaporin-2 (AQP2) antibodies. (B) Densitometric analyses revealed that AQP2 expression in inner medulla was increased in CCl4-treated rats compared with control rats. (C and D) Inner medullary collecting duct (IMCD) from control rats showed diffuse cytoplasmic labeling of AQP2 with less prominent apical plasma membrane labeling, whereas IMCD from CCl4-treated rats with cirrhosis exhibited AQP2 labeling that was localized mainly to the apical plasma membrane domains with marginal labeling of cytoplasm. (E and F) Immunocytochemical analyses revealed similar changes in the subcellular distribution of phosphorylated-AQP2 (p-AQP2) in kidney from CCl4-treated rats. There was a marked increase in apical p-AQP2 immunolabeling in CCl4-treated rats (E) compared with control rats (F).
The changes in trafficking of AQP2 and p-AQP2 were examinedby immunoperoxidase microscopy. Immunolabeling of AQP2 was seenexclusively in collecting duct principal cells. There was aprominent difference in the subcellular localization of AQP2in kidneys from CCl4-treated and control rats in the inner medullarycollecting duct. In CCl4-treated rats, AQP2 labeling was mainlyassociated with the apical plasma membrane domains with weakerlabeling intensity of cytoplasmic domains (Figure 11D). In contrast,AQP2 labeling in kidneys of control rats showed weaker labelingintensity throughout the cytoplasm with less prominent apicalplasma membrane labeling (Figure 11C). Immunohistochemistryalso revealed similar changes in the subcellular distributionof p-AQP2 in kidney from CCl4-treated rats. There was a markedincrease in apical p-AQP2 immunolabeling in CCl4-treated rats(Figure 11F) compared with control rats (Figure 11E).
This study was undertaken to elucidate renal mechanisms in thedisturbed sodium metabolism in rats with decompensated livercirrhosis induced by chronic administration of CCl4. The renalresponses for sodium retention displayed wide variations amongthe rats with liver cirrhosis. Some rats showed markedly decreasedurinary sodium excretion (sodium retaining stage, group A),whereas the others exhibited unchanged urinary sodium excretion(maintenance stage, group B) compared with controls, even thoughall CCl4-treated rats had a significant amount of ascites. Theresults demonstrated that CCl4-induced sodium retaining stage(group A) of liver cirrhosis was associated with (1) decreasedurinary sodium excretion and increased or maintained plasmaaldosterone levels; (2) increased apical targeting of ENaC subunitsin DCT2, CNT and collecting duct segments; and (3) decreasedprotein abundance of 11HSD2. In contrast, maintenance stage(group B) of liver cirrhosis was associated with no changesin the urinary sodium excretion, trafficking, and abundanceof ENaC subunits and the abundance of 11HSD2.
Increased Apical Targeting of ENaC Subunits in CCl4-Treated Rats
In this study, the most important finding is the striking increasein targeting of all ENaC subunits to the apical plasma membranedomain in DCT2, CNT, and collecting duct in the sodium-retainingstage (group A) but not in the maintenance stage (group B) ofliver cirrhosis. Because an increased targeting of all ENaCsubunits to the apical plasma membrane is associated with increasedsodium reabsorption, our finding of an increased ENaC targetingin the sodium-retaining stage of liver cirrhosis could contributesignificantly to the increased renal tubular sodium reabsorption.These observations therefore strongly support the view thatthe renal sodium retention in the decompensated liver cirrhosisis caused mainly by an increased sodium reabsorption in distalnephron, including the collecting duct (6). It is noteworthythat the experimental animals are slightly hyponatremic relativeto the controls. Thus, water retention seems to exceed the sodiumretention. Consistent with this, the changes in AQP2 all areconsistent with increased vasopressin levels (2,23). Arguably,this means that the sodium retention is appropriate to retainplasma osmolality.
Altered Protein Abundance of ENaC Subunits in CCl4-Treated Rats
We demonstrated that the abundance of -ENaC was decreased orunchanged and the abundance of the 70-kD form of -ENaC was increasedwhereas the 85-kD band was markedly decreased in the sodium-retainingstage (group A) of rats with cirrhosis rats. Previous studiesdemonstrated that aldosterone causes a mobility shift of -ENaCfrom an 85- to a 70-kD band without a change in total -ENaCprotein abundance (13). The appearance of the 70-kD form of-ENaC in response to aldosterone is putatively due to a channel-activatingproteolytic cleavage (27). The same changes are observed inchronically sodium-restricted rats in addition to a significantdownregulation of the -ENaC subunit (13). Thus, the observedincreased apical targeting and altered expression of - and -ENaCsubunits in group A could be caused by the stimulation of MRin the aldosterone-responsive epithelium.
Recent studies demonstrated that the abundance of NCC and -ENaCare increased by aldosterone treatment in normal rats (13,28).In contrast, in our study we did not observe any changes ofthe abundance of NCC and -ENaC in CCl4-induced liver cirrhosis,where plasma aldosterone levels were elevated. Consistent withthis, we previously demonstrated that puromycin aminonucleoside–inducednephrotic syndrome was associated with an increased apical targetingof ENaC subunits, but the protein abundance of -ENaC was notchanged in the kidney cortex in the presence of increased plasmaaldosterone levels (24). Moreover, we previously demonstratedthat -ENaC abundance was not changed in lithium-treated rats,in which plasma aldosterone levels were significantly increased(29). In both puromycin aminonucleoside–and lithium-treatedrats, apical trafficking of ENaC subunits was markedly increased,whereas NCC abundances were decreased, suggesting that changesof trafficking of ENaC subunits and abundance of aldosterone-sensitivetransporters (-ENaC and NCC) are dissociated in pathophysiologicconditions. Moreover, mineralocorticoid activity can be regulatedby different mechanisms: At the prereceptor level (e.g., 11HSD2),at the receptor level, and at the postreceptor level of transcriptionalactivation or repression by cell-specific co-factors (30). Eachof these cellular events ultimately will influence the natureand/or the magnitude of the response of the tissue after thestimulation of MR. Thus, it can be speculated that other factorsthat are at least as effective as aldosterone in modulatingENaC expression/trafficking play a role, or probably the interactionof MR is modified by some local factors in experimental livercirrhosis.
Decreased Urinary Na/K Ratio in Group A but not in Group B of Liver Cirrhosis
The urinary Na/K ratio has widely been used to evaluate aldosteroneactivity at the distal nephron and collecting duct (31,32).Accordingly, the sodium-retaining stage (group A rats with cirrhosis)in both protocols 1 and 2 was associated with markedly decreasedurinary Na/K ratio, indicating an increased aldosterone effectin the distal nephron. In contrast, maintenance stage (groupB rats with cirrhosis) in protocols 1 and 2 was associated withunchanged Na/K ratio and marginally decreased plasma aldosteronelevels. This may play a compensatory role to promote the urinarysodium excretion in this stage of liver cirrhosis. Thus, dynamicchanges of circulating aldosterone levels could play a rolein the sodium retention in liver cirrhosis.
At the initial stage of decompensated cirrhosis, in which therenin-angiotensin-aldosterone system (RAAS) and sympatheticnervous system are usually not activated, sodium retention couldbe due to mechanisms that are unrelated to arterial vascularunderfilling and increased plasma aldosterone levels (33,34).This would correspond to what we observed in 11 wk protocol.Circulatory dysfunction at this phase, although greater thanin compensated cirrhosis without ascites, is not intense enoughto stimulate the RAAS (35). However, previous studies on theintrarenal sodium handling strongly indicate that sodium retentionobserved in this stage occurs predominantly at the aldosterone-sensitivedistal nephron (7). Thus, in addition to the action of aldosteroneitself, other possible mechanisms contributing to the activationof MR at the distal nephron should be considered. As diseaseprogresses, an activation of RAAS and sympathetic nervous systemcould also be involved in the pathogenesis of sodium retentionlater on, and this results in a more severe impairment in thesodium and water balance (33,34). In this study, we demonstratedthat in the sodium-retaining stage (group A) of liver cirrhosis,an increased urinary sodium reabsorption was associated withthe markedly increased apical targeting of ENaC subunits aswell as decreased urinary Na/K ratio with maintained (11-wkexperiment) or increased (12-wk experiment) plasma aldosteronelevels. These differences may verify that the pathophysiologicstate reached in the two phases is different even though bothare associated with sodium retention.
Decreased Protein Abundance and Immunolabeling Intensity of 11HSD2 in CCl4-Treated Rats with Sodium Retention
We also demonstrated that there is a downregulation of 11HSD2in renal cortex/OSOM. This finding is consistent with recentstudies that demonstrated decreased 11HSD2 activity in the kidneysof liver cirrhosis induced by bile duct obstruction (35,36).These findings suggest that reduced activity of 11HSD2 providesunhindered access of glucocorticoids to the MR, resulting inthe increased aldosterone effectiveness and renal sodium retention.In this study, we demonstrated that an increased apical targetingof ENaC subunits was associated with the decreased protein abundanceof 11HSD2 in the sodium-retaining stage of liver cirrhosis (groupA) in both protocols 1 and 2, in which urinary sodium excretionwas markedly decreased. Furthermore, a mobility shift of -ENaCfrom an 85 to a 70-kD band, a representative finding of aldosteroneeffect, was also associated with decreased 11HSD2 abundance.Thus, increased apical targeting and changes of ENaC abundancemay be attributed partly to the increased MR activity causedby decreased abundance of 11HSD2.
Plasma aldosterone levels have been shown to be reduced in conditionswith low expression of 11HSD2 in normal human or rat (21,37).In our study, however, plasma aldosterone levels were not reducedin the sodium-retaining stage of liver cirrhosis (group 1) despitethat the protein abundance of 11HSD2 was significantly decreased.Thus, coordinated activation of the MR by both glucocorticoidas a consequence of reduced activity of 11HSD2 and increasedplasma aldosterone level may stimulate the distal renal tubularsodium reabsorption and potassium loss.
Increased Abundances of NHE3 and NKCC2 in ISOM from CCl4-Treated Rats
It has been demonstrated that rats with common bile duct ligation–inducedliver cirrhosis had an increased natriuretic response to furosemidetogether with marked hypertrophy of the mTAL cells in the ISOM(38,39). During the later decompensated stage, in which therenal sympathetic nerves and the RAAS are activated, cirrhosisis associated with avid sodium retention and edema. Concomitantincrease of plasma vasopressin, glucagon, and insulin also contributeto the stimulation of sodium reabsorption in the mTAL in ratswith cirrhosis (40–42). In this study, we demonstratedthe increased abundance of NHE3 and NKCC2 in the ISOM in CCl4-treatedrats. These observations therefore support the view that theincreased renal sodium reabsorption associated with the latedecompensated stage of cirrhosis is caused partly by the increasedsodium reabsorption in mTAL (43). We here suggest that thisoccurs via upregulated protein abundance of NHE3 and NKCC2 inthe mTAL.
Increased Protein Abundance and Apical Targeting of AQP2 and p-AQP2
Regarding the involvement of AQP2 in water retention in livercirrhosis, previous animal studies showed conflicting results.It was demonstrated that CCl4-induced liver cirrhosis in ratsresulted in increased or unchanged expression of AQP2 mRNA andprotein (23,44–46). This discrepancy of AQP2 expressionamong the studies with the CCl4 model of decompensated livercirrhosis is not well understood. In the 12-wk experiment ofour study, the protein abundance and apical targeting of AQP2and p-AQP2 were increased in the inner medulla, along with theincreased urine osmolality and urine to plasma osmolality ratioin CCl4-treated rats. In addition, plasma osmolality was significantlydecreased and CCl4-treated rats were marginally hyponatremiccompared with controls. These findings indicate that upregulationof the vasopressin-regulated water channel AQP2 abundance maycontribute to the increased renal water reabsorption in decompensatedliver cirrhosis. In accordance with our results, a previousstudy that used differential centrifugation of kidney proteinsamples in rats with liver cirrhosis showed evidence for theredistribution of AQP2 to the plasma membrane (46). Consistentwith this, urinary AQP2 excretion, which would be attributedto the increased cellular trafficking, was increased in patientswith liver cirrhosis (47). Thus, the inconsistent findings ofAQP2 abundance in kidneys of animals with liver cirrhosis mayreflect the variation of disease severity.
Conclusion
The results demonstrate underlying molecular mechanisms forthe disturbed sodium metabolism in rats with decompensated livercirrhosis induced by chronic administration of CCl4. Increasedapical targeting of ENaC subunits combined with diminished abundanceof 11HSD2 in the DCT2, CNT, and collecting duct is likely toplay an additive role in the sodium-retaining stages of livercirrhosis. The upregulation of the vasopressin-regulated waterchannel AQP2 as well as increased apical AQP2 targeting is likelyto contribute to the increased water reabsorption and urinaryconcentration in liver cirrhosis.
Acknowledgments
We thank Ida Maria Jalk, Gitte Kall, Helle Høyer, ZhilaNikrozi, Inger Merete Paulsen, Lotte Vallentin Holbech, MetteVistisen, and Dorte Wulff for expert technical assistance. TheWater and Salt Research Center at the University of Aarhus isestablished and supported by the Danish National Research Foundation(Danmarks Grundforskningsfond). Support for this study was providedby the Karen Elise Jensen Foundation, Human Frontier ScienceProgram; the European Commission (QRLT 2000 00778 and QRLT 200000987); the Regional Technology Innovation Program of the MOCIE(RTI04-01-01, T.-H.K.); and the intramural budget of the NationalHeart, Lung, and Blood Institute, National Institutes of Health.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication August 31, 2004.
Accepted for publication August 9, 2005.
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[Abstract][Full Text][PDF]
-Hydroxysteroid Dehydrogenase in Rats with CCl4-Induced Decompensated Liver Cirrhosis
Top
Abstract
Introduction
-hydroxysteroid dehydrogenase (11
-ENaC was unchanged, whereas 
-ENaC underwent a complex change associated with increased abundance of the 70-kD band with a concomitant decrease in the main 85-kD band, corresponding to an aldosterone effect. In contrast, no changes in the abundance of ENaC subunit were observed in group B. Immunoperoxidase microscopy revealed an increased apical targeting of 
; group A) of the rats with CCl4-induced cirrhosis showed markedly decreased urinary sodium excretion, whereas the other six rats (
; group B) remained similar compared with controls. (B) The urinary Na/K ratio was decreased in rats with cirrhosis, indicating increased aldosterone effectiveness in the distal nephron. *P < 0.05 versus control.
