Biphasic changes of epithelial sodium channel abundance and trafficking in common bile duct ligation-induced liver cirrhosis
Authors: S.W. Kim, W. Wang, M.C. Sassen, K.C. Choi, J.S. Han, M.A. Knepper, TEN Jonassen, J. Frøkiær, S. Nielsen
Journal: Kidney International (2005)

We hypothesize that dysregulation of the epithelial sodium channel (ENaC) may be responsible for the increased sodium retention in liver cirrhosis. Liver cirrhosis was induced by common bile duct ligation (CBDL). We examined the abundance of ENaC subunits and type 2 isoform of 11β-hydroxysteroid dehydrogenase (11βHSD2) in the kidney by immunoblotting and immunohistochemistry at 6 or 8 weeks after operation. At 6 weeks, cirrhotic rats had developed ascites and displayed a positive sodium balance. The urinary sodium excretion and fractional excretion of sodium were decreased, while plasma aldosterone was unchanged. The abundance of ENaC subunits was not changed in the cortex and outer stripe of the outer medulla (OSOM). In contrast, immunoperoxidase microscopy revealed an increased apical targeting of α-, β- and γENaC in late distal convoluted tubule, connecting tubule and collecting duct. Moreover, 11βHSD2 abundance was decreased in the cortex/OSOM and inner stripe of the outer medulla. At 8 weeks, urinary sodium excretion and fractional excretion of sodium were not changed, while the plasma aldosterone level was decreased. The expression of ENaC subunits was decreased in the cortex/OSOM. Immunoperoxidase microscopy confirmed decreased expression of ENaC subunits, whereas subcellular localization was not changed. These results suggest that increased apical targeting of ENaC subunits and diminished abundance of 11βHSD2 may contribute to promote sodium retention in the sodium-retaining stage of liver cirrhosis (at 6 weeks). The subsequent decreased expression and reduced targeting of ENaC subunits may play a role in promoting sodium excretion in the later stage of liver cirrhosis (at 8 weeks). We hypothesize that dysregulation of the epithelial sodium channel (ENaC) may be responsible for the increased sodium retention in liver cirrhosis. Liver cirrhosis was induced by common bile duct ligation (CBDL). We examined the abundance of ENaC subunits and type 2 isoform of 11β-hydroxysteroid dehydrogenase (11βHSD2) in the kidney by immunoblotting and immunohistochemistry at 6 or 8 weeks after operation. At 6 weeks, cirrhotic rats had developed ascites and displayed a positive sodium balance. The urinary sodium excretion and fractional excretion of sodium were decreased, while plasma aldosterone was unchanged. The abundance of ENaC subunits was not changed in the cortex and outer stripe of the outer medulla (OSOM). In contrast, immunoperoxidase microscopy revealed an increased apical targeting of α-, β- and γENaC in late distal convoluted tubule, connecting tubule and collecting duct. Moreover, 11βHSD2 abundance was decreased in the cortex/OSOM and inner stripe of the outer medulla. At 8 weeks, urinary sodium excretion and fractional excretion of sodium were not changed, while the plasma aldosterone level was decreased. The expression of ENaC subunits was decreased in the cortex/OSOM. Immunoperoxidase microscopy confirmed decreased expression of ENaC subunits, whereas subcellular localization was not changed. These results suggest that increased apical targeting of ENaC subunits and diminished abundance of 11βHSD2 may contribute to promote sodium retention in the sodium-retaining stage of liver cirrhosis (at 6 weeks). The subsequent decreased expression and reduced targeting of ENaC subunits may play a role in promoting sodium excretion in the later stage of liver cirrhosis (at 8 weeks). Liver cirrhosis is a chronic disease with marked progressive changes in systemic and renal hemodynamics. Initially, liver cirrhosis patients have peripheral vasodilatation and increased cardiac output but do not have clinical signs of fluid retention (the compensated state). During the late decompensated state, liver cirrhosis is associated with sodium retention, edema and ascites. Sodium retention is the most frequent renal functional abnormality in patients and animal models with liver cirrhosis.1.Cardenas A. Bataller R. Arroyo V. Mechanisms of ascites formation.Clin Liver Dis. 2000; 4: 447-465Google Scholar However, the mechanism for the sodium retention is still incompletely understood and the molecular basis remains undefined. As sodium retention is usually associated with a marked activation of the renin–aldosterone system and can be reversed by spironolactone, a specific antagonist of the tubular effect of aldosterone,2.Jimenez W. Martinez-Pardo A. Arroyo V. et al.Temporal relationship between hyperaldosteronism, sodium retention and ascites formation in rats with experimental cirrhosis.Hepatology. 1985; 5: 245-250Google Scholar it has been suggested that hyperaldosteronism is among the most important mechanisms involved in the avid sodium retention in patients with decompensated liver cirrhosis. However, evidence provided by several studies indicates that the extent of sodium retention and potassium loss cannot be explained completely by the elevated aldosterone concentrations in cirrhotic patients, and renal sodium retention precedes ascites formation.2.Jimenez W. Martinez-Pardo A. Arroyo V. et al.Temporal relationship between hyperaldosteronism, sodium retention and ascites formation in rats with experimental cirrhosis.Hepatology. 1985; 5: 245-250Google Scholar, 3.Levy M. Allotey J.B. Temporal relationships between urinary salt retention and altered systemic hemodynamics in dogs with experimental cirrhosis.J Lab Clin Med. 1978; 92: 560-569Google Scholar, 4.Wensing Q. Sabra R. Branch R.A. Renal and systemic hemodynamics in experimental cirrhosis in rats: relation to hepatic function.Hepatology. 1990; 12: 13-19Google Scholar Previous findings indicate that aldosterone may be normal in a significant proportion of patients with ascites.5.Perez-Ayuso R.M. Arroyo V. Planas R. et al.Randomized comparative study of efficacy of furosemide versus spironolactone in nonazotemic cirrhosis with ascites. Relationship between the diuretic response and the activity of the renin–aldosterone system.Gastroenterology. 1983; 84: 961-968Scopus (274) Google Scholar Although plasma aldosterone levels are not increased, it is well known that treatment with mineralocorticoid receptor (MR) antagonists can prevent sodium retention in animal models of cirrhosis2.Jimenez W. Martinez-Pardo A. Arroyo V. et al.Temporal relationship between hyperaldosteronism, sodium retention and ascites formation in rats with experimental cirrhosis.Hepatology. 1985; 5: 245-250Google Scholar, 6.Jonassen T.E. Petersen J.S. Sorensen A.M. et al.Aldosterone receptor blockade inhibits increased furosemide-sensitive sodium reabsorption in rats with liver cirrhosis.J Pharmacol Exp Ther. 1998; 287: 931-936Google Scholar and, clinically, aldosterone antagonists are the established first choice in the treatment of cirrhotic ascites and edema.7.Gines P. Cardenas A. Arroyo V. Rodes J. Management of cirrhosis and ascites.N Engl JMed. 2004; 350: 1646-1654Google Scholar It has therefore been suggested that the sensitivity of the collecting duct to aldosterone is increased in liver cirrhosis. Consistent with this, the activity of type 2 11β-hydroxysteroid dehydrogenase (11βHSD2), an enzyme which normally protects the MR from stimulation by glucocorticoids, is significantly decreased in microdissected cortical collecting tubules from rats with liver cirrhosis induced by common bile duct ligation (CBDL).8.Ackermann D. Vogt B. Escher G. et al.Inhibition of 11 beta-hydroxysteroid dehydrogenase by bile acids in rats with cirrhosis.Hepatology. 1999; 30: 623-629Google Scholar The occupancy of MR by glucocorticoid hormone will lead to permanent maximal sodium retention. If this mechanism is operative in rats with liver cirrhosis, collecting duct sodium reabsorption might well be stimulated even in the presence of normal plasma levels of aldosterone. As the epithelial sodium channel (ENaC) mediates the major sodium transport in the collecting duct9.Duc C. Farman N. Canessa C.M. et al.Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.J Cell Biol. 1994; 127: 1907-1921Google Scholar and both protein abundance and apical membrane targeting of ENaC are regulated by hormones such as aldosterone10.Masilamani S. Kim G.H. Mitchell C. et al.Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.J Clin Invest. 1999; 104: R19-R23Google Scholar and vasopressin,11.Ecelbarger C.A. Kim G.H. Terris J. et al.Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney.Am J Physiol Renal Physiol. 2000; 279: F46-F53Google Scholar we speculate that altered expression and/or apical membrane targeting of ENaC subunits (α, β and γ) may account for the increased sodium absorption in the distal nephron and the collecting ducts in liver cirrhosis. The purpose of this study was therefore to investigate whether CBDL-induced decompensated liver cirrhosis in rats is associated with altered regulation of ENaC subunit protein abundance and/or changes in the segmental and subcellular localization. We established a window at 6 weeks after bile duct ligation as a critical time point at which ascites develops in rats. At 6 weeks, CBDL-induced cirrhotic rats developed significant ascites, and exposure of the abdomen at the termination of the study showed that the cirrhotic rats had a large amount of ascites. After 6 weeks, the quantity of peritoneal fluid in cirrhotic rats was significantly greater than in controls. Plasma concentrations of alanine aminotransaminase (ALAT) and bilirubin were significantly increased in cirrhotic rats. Plasma creatinine levels were not changed, whereas renal creatinine clearance was significantly decreased in rats with CBDL. Importantly, the 24-h urinary sodium excretion and fractional excretion of sodium were significantly decreased at 6 weeks in rats with CBDL compared with controls. Consequently, cirrhotic rats displayed a positive sodium balance. The urinary Na/K ratio was markedly decreased in 6-week CBDL rats, indicating increased aldosterone effectiveness in the distal nephron. Accordingly, fractional excretion of potassium was increased. On the other hand, plasma aldosterone levels were not changed in rats with 6-week CBDL.Table 1Changes in hepatic and renal function in control rats and rats with 6-week CBDLControl (n=7)Cirrhosis (n=10)Body weight (g)312±2295±8Food intake (g)16.7±0.513.7±1.9Water intake (ml)23.4±0.621.9±2.5Ascites (ml)Nondetectable4.5±1.2**UO (μl/min)9.8±0.711.1±1.0PALAT (U/l)38.7±1.864.1±4.6**Pbilurubin (μmol/1)1.8±0.1104.9±5.2**Pcr (μmol/1)34.1±1.436.4±2.2Ccr (ml/min)1.51±0.061.11±0.12*PNa (mEq/1)138.6±0.5139.6±0.7PK (mEq/l)5.5±0.14.6±0.2**UNa × UO (mmol)1.27±0.080.63±0.11**UK × UO (mmol)4.19±0.103.39±0.39Sodium balance (mmol)0.26±0.070.52±0.05*FENa (%)0.42±0.020.28±0.05*FEK (%)35.5±1.047.6±3.6**Urine Na/K0.30±0.010.19±0.03**Paldosterone (pg/ml)339±53357±53Uosm (mosm/kgH2O)1826±1031528±171Posm (mosm/kgH2O)298±1298±1U/Posm6.1±0.45.1±0.6Values are expressed as mean±s.e. These values are measured at the last day of experiments (6 weeks). UO, urine output; P alat, plasma alanine aminotransferase; Pbilurubin, plasma bilirubin; PNa, plasma sodium; PK, plasma potassium; Pcr, plasma creatinine; Ccr, creatinine clearance; UNa × UO, rate of urinary sodium excretion; UK × UO, rate of urinary potassium excretion; sodium balance, the difference between dietary sodium intake and urinary sodium excretion in the 24 h; FENa, fractional excretion of sodium in urine; FEK, fractional excretion of potassium in urine; urine Na/K, urine sodium/potassium ratio; Paldosterone, plasma aldosterone; Uosm, urine osmolality; Posm, plasma osmolality. *P<0.05, **P<0.01 compared with control. Open table in a new tab Values are expressed as mean±s.e. These values are measured at the last day of experiments (6 weeks). UO, urine output; P alat, plasma alanine aminotransferase; Pbilurubin, plasma bilirubin; PNa, plasma sodium; PK, plasma potassium; Pcr, plasma creatinine; Ccr, creatinine clearance; UNa × UO, rate of urinary sodium excretion; UK × UO, rate of urinary potassium excretion; sodium balance, the difference between dietary sodium intake and urinary sodium excretion in the 24 h; FENa, fractional excretion of sodium in urine; FEK, fractional excretion of potassium in urine; urine Na/K, urine sodium/potassium ratio; Paldosterone, plasma aldosterone; Uosm, urine osmolality; Posm, plasma osmolality. *P<0.05, **P<0.01 compared with control. The protein abundance of αENaC was not changed in the cortex/outer stripe of the outer medulla (OSOM), while it was increased in the inner stripe of the outer medulla (ISOM) in rats with 6-week CBDL compared with controls (Figure 1 ). The abundance of βENaC was not changed in the cortex/OSOM and ISOM. The 85 kDa band abundance of γENaC was decreased in the cortex/OSOM (Figure 1, Table 2 ), but not in the ISOM. However, total γENaC abundance and 70 kDa band expression were not changed in the cortex/OSOM and ISOM. The analyses of normalized band densities are shown in Table 2.Table 2Densitometric analysis of ENaC expression in control rats and rats with 6-week CBDLControlCirrhosis(n=7) (%)(n=10) (%)Cortex/OSOM αENaC100±10136±29 βENaC100±1793±32 γENaC (total)100±682±12 γENaC 85 kDa100±365±7* γENaC 70 kDa100±27118±40ISOM αENaC100±12154±10* βENaC100±1773±37 γENaC (total)100±7117±13 γENaC 85 kDa100±584±7 γENaC 70 kDa100±18247±102Values are expressed as mean±s.e. ENaC, epithelial sodium channel; OSOM, outer stripe of outer medulla; ISOM, inner stripe of outer medulla. *P<0.05 compared with control. Open table in a new tab Values are expressed as mean±s.e. ENaC, epithelial sodium channel; OSOM, outer stripe of outer medulla; ISOM, inner stripe of outer medulla. *P<0.05 compared with control. In control rats, immunoperoxidase staining for the βENaC subunits showed diffuse punctuate labeling throughout the distal convoluted tubule (DCT2), connecting tubule (CNT) and collecting duct principal cells. At 6-weeks in CBDL rats, βENaC labeling was predominantly localized to the apical domains (i.e. plasma membrane and intracellular vesicles close to the membrane), with only marginal labeling of the cytoplasm corresponding to labeling of intracellular vesicles. This increased apical targeting was evident in almost all cross-sectioned tubules of DCT2 (not shown), CNT (Figure 2a and b ), cortical collecting duct (CCD, Figure 2c and d) and outer medullary collecting duct (OMCD) (not shown). Moreover, immunohistochemical analysis also revealed similar changes in the subcellular distribution of γENaC in kidneys from 6-week CBDL rats. There was a marked increase in apical γENaC immunolabeling in DCT2 (not shown), CNT (Figure 2e and f), CCD (Figure 2g and h) and OMCD (not shown) in kidneys from 6-week CBDL rats. Finally, immunoperoxidase microscopy of sections from kidneys also revealed increased apical immunolabeling of αENaC as well in DCT2 (not shown), CNT (not shown), CCD (Figure 3a and b ) and OMCD (Figure 3c and d) in 6-week CBDL rats.Figure 3Immunoperoxidase microscopy of αENaC in CCD and OMCD. Immunoperoxidase labeling of αENaC is restricted to a narrow zone in the apical part including the plasma membrane domains of the principal cells of the CCD (a) and OMCD (c) in control rats. In contrast, rats with 6-week CBDL revealed markedly increased apical immunolabeling of αENaC in CCD (b) and OMCD (d). Arrows indicate apical labeling. Original Magnification: × 630.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The protein abundance of 11βHSD2 was significantly decreased in the cortex/OSOM (40±11 vs 100±14%, P<0.05, Figure 4a and b) and ISOM (71±10 vs 100±9%, P<0.05). Immunoperoxidase microscopy also demonstrated that the immunolabeling of 11βHSD2 was markedly decreased in CNT (Figure 4c and d) and CCD (Figure 4e and f). The protein expression of α1-isoform of Na,K-ATPase was decreased in the cortex/OSOM, but not in the ISOM. Immunoblotting analysis revealed that type 3 Na/H exchanger (NHE3) expression was decreased in the cortex/OSOM, but not in the ISOM. Immunocytochemistry demonstrated that the labeling of NHE3 in the proximal tubule in rats with 6-week CBDL was reduced, but maintained in the medullary thick ascending limb (TAL) compared with control rats (not shown). Immunoblotting analysis revealed a decreased expression of Na–K-2Cl cotransporter (NKCC2) in the cortex/OSOM in 6-week cirrhotic rats whereas it was not altered in the ISOM. In contrast to the significant changes in renal protein expressions of NHE3 and NKCC2 in rats with 6-week CBDL, immunoblotting analysis revealed unchanged levels of Na–Cl cotransporter (NCC) in the cortex/OSOM.Table 3Densitometric analysis of renal sodium transporter expression in control rats and rats with 6-week CBDLControlCirrhosis(n=7) (%)(n=10) (%)Cortex/OSOM Na,K-ATPase α1100±1156±6* NHE3100±1132±13* NKCC2100±1261±6* NCC100±883±5ISOM Na,K-ATPase α1100±9104±4 NHE3100±24107±16 NKCC2100±12105±6Values are expressed as mean±s.e. NHE3, type 3 Na/H exchanger; NKCC2, Na-K-2Cl cotransporter; NCC, Na–Cl cotransporter. *P<0.05 compared with control. Open table in a new tab Values are expressed as mean±s.e. NHE3, type 3 Na/H exchanger; NKCC2, Na-K-2Cl cotransporter; NCC, Na–Cl cotransporter. *P<0.05 compared with control. At 8 weeks, all CBDL rats developed significant ascites. Plasma concentrations of ALAT and bilirubin were significantly increased in rats with 8-week CBDL compared with controls. Plasma creatinine levels were not changed, whereas renal creatinine clearance was significantly decreased in rats with liver cirrhosis. In contrast to reduced sodium excretion in rats with 6-week CBDL, the 24-h urinary sodium excretion and fractional excretion of sodium were not changed at 8-week CBDL. Consequently, liver cirrhosis rats did not show any changes in sodium balance, and the urinary Na/K ratio and fractional excretion of potassium were also normal. Interestingly, plasma aldosterone levels were significantly decreased in 8-week CBDL.Table 4Changes in hepatic and renal function in control rats and rats with 8-week CBDLControlCirrhosis(n=7)(n=8)Body weight (g)302±4300±15Food intake (g)19.0±0.217.8±1.4Water intake (ml)26.7±1.524.1±0.9Ascites (ml)Nondetectable8.5±2.0**UO (μl/min)13.3±1.411.5±1.0Palat (U/l)49.8±1.595.0±7.8**Pbilurubin (μmol/1)2.1±0.1101.8±11.5**Pcr (μmol/l)29.7±2.626.8±2.2Ccr (ml/min)1.57±0.091.26±0.08*PNa (mEq/1)138.3±0.4139.3±0.6PK (mEq/l)4.97±0.265.36±0.17UNa × UO (mmol)1.57±0.071.19±0.16UK × UO (mmol)4.16±0.143.47±0.14**Sodium balance, (mmol)0.23±0.070.33±0.11FENa (%)0.52±0.040.47±0.06FEK (%)37.7±1.736.6±2.0Urine Na/K0.38±0.010.34±0.04Paldosterone (pg/ml)346±58181±7*Uosm (mosm/kgH2O)1615±1761764±194Posm (mosm/kgH2O)300±1300±3U/Posm5.4±0.65.9±0.7Values are expressed as mean±s.e. These values are measured on the last day of experiments (8 weeks). UO, urine output; P alat, plasma alanine aminotransferase; Pbilurubin, plasma bilirubin; PNa, plasma sodium; PK, plasma potassium; Pcr, plasma creatinine; Ccr, creatinine clearance; UNa × UO, rate of urinary sodium excretion; UK × UO, rate of urinary potassium excretion; sodium balance, the difference between dietary sodium intake and urinary sodium excretion in the 24 h; FENa, fractional excretion of sodium into urine; FEK, fractional excretion of potassium in urine; urine Na/K, urine sodium/potassium ratio; Paldosterone, plasma aldosterone; Uosm, urine osmolality; Posm, plasma osmolality. *P<0.05, **P<0.01 compared with control. Open table in a new tab Values are expressed as mean±s.e. These values are measured on the last day of experiments (8 weeks). UO, urine output; P alat, plasma alanine aminotransferase; Pbilurubin, plasma bilirubin; PNa, plasma sodium; PK, plasma potassium; Pcr, plasma creatinine; Ccr, creatinine clearance; UNa × UO, rate of urinary sodium excretion; UK × UO, rate of urinary potassium excretion; sodium balance, the difference between dietary sodium intake and urinary sodium excretion in the 24 h; FENa, fractional excretion of sodium into urine; FEK, fractional excretion of potassium in urine; urine Na/K, urine sodium/potassium ratio; Paldosterone, plasma aldosterone; Uosm, urine osmolality; Posm, plasma osmolality. *P<0.05, **P<0.01 compared with control. The protein abundance of all ENaC subunits was markedly decreased in the cortex/OSOM in rats with 8-week CBDL compared with controls (Figure 6 ). The abundance of βENaC and γENaC was also decreased in the ISOM, while αENaC expression was unchanged. The analyses of normalized band densities are shown in Table 5 . Thus, these changes may promote sodium excretion in response to earlier sodium retention.Table 5Densitometric analysis of ENaC and 11βHSD2 expression in control rats and rats with 8-week CBDLControlCirrhosis(n=7) (%)(n=8) (%)Cortex/OSOM αENaC100±855±6* βENaC100±636±7* γENaC (total)100±1063±9* 11βHSD2100±737±4*ISOM αENaC100±783±17 βENaC100±1744±11* γENaC (total)100±1653±8* 11βHSD2100±479±2*Values are expressed as mean±s.e. ENaC, epithelial sodium channel; 11 βHSD2, type 2 isoform of 11β-hydroxysteroid dehydrogenase; OSOM, outer stripe of outer medulla; ISOM, inner stripe of outer medulla. *P<0.05 compared with control. Open table in a new tab Values are expressed as mean±s.e. ENaC, epithelial sodium channel; 11 βHSD2, type 2 isoform of 11β-hydroxysteroid dehydrogenase; OSOM, outer stripe of outer medulla; ISOM, inner stripe of outer medulla. *P<0.05 compared with control. The protein abundance of 11βHSD2 was decreased in the cortex/OSOM and ISOM (Figure 6, Table 5). βENaC labeling intensity was markedly decreased in the principal cells of DCT2 (not shown), CNT (Figure 7a and b ), cortical (Figure 7c and d), and OMCD (not shown), consistent with the immunoblotting analysis (Figure 6), and the labeling was mainly cytoplasmic. The immunlabeling intensity of γENaC was significantly decreased in DCT2 (not shown), CNT (Figure 7e and f), cortical (Figure 7g and h) and OMCD (not shown) in rats with 8-week cirrhosis compared with controls, whereas the subcellular localization did not change. The αENaC labeling was significantly decreased in DCT2 (not shown), CNT (Figure 7i and j) and the collecting duct (not shown) in rats with 8-week cirrhosis compared with controls. The protein abundance of NHE3 was decreased in the cortex/OSOM, while it was unchanged in the ISOM. In contrast, the protein abundance of NCC, NKCC2 and α1 subunit of Na, K-ATPase was not changed in the cortex/OSOM or ISOM in rats with 8-week CBDL (Figure 8 , Table 6 ). Immunocytochemistry also revealed that the labeling of NHE3 in the proximal tubule was reduced, but was unchanged in TAL in rats with 8-week CBDL compared with control rats (Figure 9 ). This is consistent with NHE3 protein abundance in the cortex/OSOM, and unchanged NHE3 expression in the ISOM as observed by immunoblotting.Table 6Densitometric analysis of renal sodium transporter expression in control rats and rats with 8-week CBDLControlCirrhosis(n=7%)(n=8%)Cortex/OSOM Na,K-ATPase α1100±886±5 NHE3100±869±8* NKCC2100±18118±9 NCC100±8110±14ISOM Na,K-ATPase α1100±496±7 NHE3100±17104±14 NKCC2100±992±14Values are expressed as mean±s.e. NHE3, type 3 Na/H exchanger; NKCC2, Na-K-2Cl cotransporter; NCC, Na-Cl cotransporter. *P<0.05 compared with control. Open table in a new tab Figure 9Immunoperoxidase microscopy of NHE3 in the proximal tubule and thick ascending limb. In controls, NHE3 labeling was seen in apical plasma membrane domains of proximal tubule cells in the cortex (a) and TAL in the ISOM (c). In rats with 8-week CBDL, the labeling of NHE-3 in the proximal tubule was reduced (b) but not in TAL (d) compared with control rats. G, glomerulus; PT, proximal tubule; TAL, thick ascending limb. Original Magnification: × 250.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Values are expressed as mean±s.e. NHE3, type 3 Na/H exchanger; NKCC2, Na-K-2Cl cotransporter; NCC, Na-Cl cotransporter. *P<0.05 compared with control. Long-term bile duct ligation in rats is a well-known experimental model of liver cirrhosis and portal hypertension, and has been widely used to study the renal mechanisms of sodium retention.12.Kountouras J. Billing B.H. Scheuer P.J. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat.Br J Exp Pathol. 1984; 65: 305-311Google Scholar In the present study, CBDL rats showed decreased fractional excretion of sodium and hence, positive sodium balance in the sodium-retaining stage of liver cirrhosis (at 6 weeks). It is suggested that increased renal tubular absorption of sodium may account for the sodium retention and pathogenesis of ascites formation. It is noteworthy that at 8 weeks of CBDL, we did not see any evidence of positive sodium balance even though all rats showed marked ascites. These findings may suggest that there are dynamic changes in sodium retention at different stages (i.e., a sodium-retaining stage and a compensatory stage) of liver cirrhosis. It should be emphasized that the two time periods are two in a continuum of changes during the development of sodium retention and the stages with partial or full compensation. In the present study, the most important finding is the increased targeting of all ENaC subunits to the apical plasma membrane domain in DCT2, CNT and collecting duct in the sodium-retaining cirrhotic rats, but not in 8-week CBDL rats without positive sodium balance. Because increased ENaC plasma membrane targeting is strongly associated with increased sodium reabsorption, this finding very strongly suggests that increased ENaC targeting contributes significantly to the increased renal tubular sodium reabsorption. Previous studies demonstrate that aldosterone causes a dramatic redistribution of ENaC to the apical plasma membrane.10.Masilamani S. Kim G.H. Mitchell C. et al.Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.J Clin Invest. 1999; 104: R19-R23Google Scholar, 13.Loffing J. Zecevic M. Feraille E. et al.Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.Am J Physiol Renal Physiol. 2001; 280: F675-F682Google Scholar Thus, the increased apical targeting of ENaC subunits in CBDL-induced liver cirrhosis may be caused by increased aldosterone activity via stimulation of the MR in the aldosterone responsive epithelium. As there was a markedly enhanced apical plasma membrane expression of all ENaC subunits at 6-week CBDL associated with maintained plasma aldosterone levels but not at 8-week CBDL when aldosterone levels were markedly reduced, it is likely that aldosterone plays an essential role. However, other mechanisms may also contribute as discussed below. In vitro studies have demonstrated that the MR has an equal affinity for aldosterone and glucocorticoids,14.Sheppard K.E. Funder J.W. Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies.J Steroid Biochem. 1987; 28: 737-742Google Scholar yet, in vivo, it displays specificity for aldosterone in the face of much higher circulating concentrations of glucocorticoids. This specificity is conferred by 11βHSD2, an enzyme that converts glucocorticoids to inactive 11-ketosteroid derivatives that have weak or no affinity for the MR in mineralocorticoid-sensitive tissues including the distal nephron.15.Funder J.W. Pearce P.T. Smith R. Smith A.I. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated.Science. 1988; 242: 583-585Google Scholar In the present study, we demonstrate that there is a downregulation of 11βHSD2 abundance in the kidney in decompensated liver cirrhotic rats. This finding is in line with recent studies in compensated liver cirrhosis reported by Quattropani et al.16.Quattropani C. Vogt B. Odermatt A. et al.Reduced activity of 11 beta-hydroxysteroid dehydrogenase in patients with cholestasis.J Clin Invest. 2001; 108: 1299-1305Google Scholar and Escher et al.17.Escher G. Nawrocki A. Staub T. et al.Down-regulation of hepatic and renal 11 beta-hydroxysteroid dehydrogenase in rats with liver cirrhosis.Gastroenterology. 1998; 114: 175-184Google Scholar They demonstrated that 11βHSD2 activity in the kidney was reduced in liver cirrhosis following bile duct ligation.16.Quattropani C. Vogt B. Odermatt A. et al.Reduced activity of 11 beta-hydroxysteroid dehydrogenase in patients with cholestasis.J Clin Invest. 2001; 108: 1299-1305Google Scholar, 17.Escher G. Nawrocki A. Staub T. et al.Down-regulation of hepatic and renal 11 beta-hydroxysteroid dehydrogenase in rats with liver cirrhosis.Gastroenterology. 1998; 114: 175-184Google Scholar These findings together suggest that reduced expression and activity provides a basis for access of glucocorticoids to the MR, thereby promoting increased collecting duct sodium reabsorption despite normal plasma levels of aldosterone in rats with experimental liver cirrhosis. Plasma aldosterone levels, at least in humans and rats without liver cirrhosis, have been shown to be reduced in conditions with low expression of 11βHSD2.18.Card W.I. Mitchell W. Strong J.A. et al.Effects of liquorice and its derivatives on salt and water metabolism.Lancet. 1953; 1: 663-668Google Scholar, 19.Turpie A.G. Thomson T.J. Carbenoxolone sodium in the treatment of gastric ulcer with special reference to side-effects.Gut. 1965; 6: 591-594Google Scholar, 20.White P.C. Mune T. Agarwal A.K. 11 beta-hydroxysteroid dehydrogenase and the syndrome of apparent mineralocorticoid excess.Endocr Rev. 1997; 18: 135-156Google Scholar It is noteworthy that plasma aldosterone levels were not reduced in rats with 6-week CBDL, despite the protein abundance of 11βHSD2 being significantly decreased. Thus, coordinated activation of MR by both glucocorticoid as a consequence of reduced activity of 11βHSD2 and by relatively high plasma aldosterone may enhance distal renal tubular sodium reabsorption and potassium loss, and hence play important roles for the pathogenesis of sodium retention and possible ascites formation in CBDL-induced liver cirrhosis. In contrast, in response to CBDL for 8 weeks, ENaC abundance was decreased and the reduction was associated with decreased plasma aldosterone levels. In addition, the increased apical targeting of ENaC subunits was retrieved in spite of decreased abundance of 11βHSD2. It can be speculated that 11βHSD2 is still functioning but not at full power (thus aldosterone may play a major role), or it may be speculated that differences in ENaC trafficking between 6 and 8 weeks are independent of aldosterone and/or 11βHSD2. In normal rats, both the apical targeting and protein abundance of αENaC are increased by high-dose aldosterone treatment.10.Masilamani S. Kim G.H. Mitchell C. et al.Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.J Clin Invest. 1999; 104: R19-R23Google Scholar However, in the present study, we did not observe an increase in αENaC protein expression in the cortex/OSOM in kidneys from rats with CBDL for 6 weeks, although increased abundance of αENaC was seen in the ISOM. It may be speculated that glucocorticoids may be involved in this regulation, and if this is the case, the observations suggest that regulation of αENaC abundance is not as sensitive as the regulation of changes in the subcellular localization of ENaC to MR activation by glucocorticoids. Whether the differential regulation of ENaC subunit targeting and expression may be related to differences in the kinetics of MR-ligand interaction remains unknown; aldosterone dissociates more slowly from MR than cortisol does, indicating that the stability of the aldosterone–MR complex is higher than that of the cortisol–MR complex.21.Hellal-Levy C. Fagart J. Souque A. Rafestin-Oblin M.E. Mechanistic aspects of mineralocorticoid receptor activation.Kidney Int. 2000; 57: 1250-1255Google Scholar, 22.Lombes M. Kenouch S. Souque A. et al.The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 11 beta-hydroxysteroid dehydrogenase.Endocrinology. 1994; 135: 834-840Google Scholar In addition, the MR activity can be regulated by at the level of transcription activation or repression by cell-specific cofactors.23.Farman N. Rafestin-Oblin M.E. Multiple aspects of mineralocorticoid selectivity.Am J Physiol Renal Physiol. 2001; 280: F181-F192Google Scholar It is also interesting to note that the rats with liver cirrhosis apparently exhibited dynamic changes in sodium retention and ENaC trafficking. After 6 weeks, CBDL animals exhibited sodium retention, potassium loss and decreased urine Na/K ratio, indicating increased aldosterone-like activity. These findings can be explained in part by decreased abundance of 11βHSD2, which permits the stimulation of MR by glucocorticoid resulting in increased apical targeting of ENaC subunits. On the other hand, at 8 weeks, the rats with liver cirrhosis did not exhibit any changes in urinary sodium and potassium excretion although they all had marked ascites. The underlying mechanism for the time course changes in circulating aldosterone levels and changes in ENaC subunits trafficking remains undefined. However, it can be speculated that at later stages of liver cirrhosis the rise in extracellular fluid volume and enhanced secretion of atrial natriuretic peptide contribute to the decrease in plasma aldosterone levels and, hence, limit ENaC abundance/trafficking as atrial natriuretic peptide has been known to inhibit directly aldosterone secretion in the adrenal glands through specific atrial natriuretic peptide receptors in the zona glomerulosa.24.Vari R.C. Freeman R.H. Davis J.O. et al.Effect of synthetic atrial natriuretic factor on aldosterone secretion in the rat.Am J Physiol. 1986; 251: R48-R52Google Scholar The abundance of Na,K-ATPase α1 subunit, NHE3 and NKCC2 was either significantly decreased in the cortex/OSOM or unchanged in the ISOM in the rats with 6-week CBDL. These results are consistent with previous observations in 5-week CBDL-induced liver cirrhosis,25.Graebe M. Brond L. Christensen S. et al.Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats.Am J Physiol Renal Physiol. 2004; 286: F288-F297Google Scholar which indicate that tubular segments distal to the TAL are responsible for the increased sodium reabsorption in cirrhosis. The decreased abundance of NHE3, NKCC2 and Na,K-ATPase in the cortex/OSOM may play a compensatory role to promote sodium excretion in the sodium-retaining stage. In addition, the decrease in NHE3 and NKCC2, as observed in this study, may balance the decrease in glomerular filtration rate, thus resulting in long-term glomerulotubular balance. Recently, it has been demonstrated that there is an increase in NKCC2 expression in kidneys from rats with CCl4-induced liver cirrhosis,26.Fernandez-Llama P. Ageloff S. Fernandez-Varo G. et al.Sodium retention in cirrhotic rats is associated with increased renal abundance of sodium transporter proteins.Kidney Int. 2005; 67: 622-630Google Scholar while NKCC2 abundance was unchanged in the ISOM or decreased in the cortex in CBDL rats, as observed in this study. Consistent with this, CBDL-induced liver cirrhosis in rats has previously been shown to be associated with marked hypertrophy of the ISOM and with either increased or maintained density of NKCC2 expression per milligram total ISOM protein.27.Jonassen T.E. Nielsen S. Christensen S. Petersen J.S. Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis.Am J Physiol. 1998; 275: F216-F225Google Scholar, 28.Jonassen T.E. Brond L. Torp M. et al.Effects of renal denervation on tubular sodium handling in rats with CBL-induced liver cirrhosis.Am J Physiol Renal Physiol. 2003; 284: F555-F563Google Scholar Thus, total ISOM expression is markedly increased in both settings. The data in the present paper are fully consistent with this. In contrast to the increased ENaC trafficking, the protein abundance of NCC remained unchanged in the cortex in 6-week liver cirrhosis. These findings may suggest that the regulation of NCC protein abundance is not as sensitive as the regulation of the subcellular localization of ENaC subunits to MR activation by glucocorticoid in liver cirrhosis. It may also be speculated that decreased glomerular filtration rate also plays a role in the unchanged expression of NCC in spite of increased MR activity. The results strongly suggest that the marked increase in apical ENaC subunit targeting combined with diminished abundance of 11βHSD2 in the DCT2, CNT and collecting duct is likely to play key roles for the sodium retention associated with CBDL-induced liver cirrhosis in rats in the sodium-retaining stage (i.e. in this model 6 weeks after CBDL). The subsequent decrease in the expression of ENaC subunits and the reduced targeting after 8 weeks of CBDL may contribute to promoting sodium excretion at the later stages of liver cirrhosis.