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【摘要】 The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor N G -nitro- L -arginine methyl ester ( L -NAME) starting on the day of common bile duct ligation (CBL). Three weeks of daily sodium balance studies showed that CBL rats developed sodium retention compared with sham-operated rats and that L -NAME treatment dose dependently deteriorated cumulative sodium balance by reducing urinary sodium excretion. Five weeks after CBL, renal clearance studies were performed, followed by Western blotting of the electroneutral type 3 sodium/proton exchanger (NHE3) and the Na-K-ATPase present in proximal tubules. Untreated CBL rats showed a decreased proximal reabsorption with a concomitant reduction of NHE3 and Na-K-ATPase levels, indicating that tubular segments distal to the proximal tubules were responsible for the increased sodium reabsorption. L -NAME-treated CBL rats showed an increased proximal reabsorption measured by the lithium clearance method and showed a marked increase in NHE3 and Na-K-ATPase protein levels. Our results show that chronic L -NAME treatment exacerbates the sodium retention found in CBL rats by a significant increase in proximal tubular reabsorption.
【关键词】 common bile duct ligation type sodium/proton exhanger proximal tubular function lithium clearance
THE EARLY STATE OF COMPENSATED cirrhosis is associated with renal sodium and water retention combined with a hyperdynamic circulation, evident as systemic arterial vasodilation with decreased total peripheral resistance and increased cardiac output. In 1991, Vallance and Moncada ( 56 ) suggested that increased systemic nitric oxide (NO) formation induced by low-grade endotoxemia could be the mediator of the hyperdynamic circulation. Since then, several studies in both animals and humans have validated this hypothesis and increased NO synthesis is now believed to be one of the early pathophysiological mechanisms of cirrhosis, being essential for systemic arterial vasodilation ( 3, 7, 19, 24, 37, 38 ).
The peripheral arterial vasodilation theory stated by Schrier and co-workers ( 49 ) proposes that the vasodilation-mediated relative vascular underfilling in cirrhosis is the primary event responsible for the renal sodium and water retention via activation of baroreceptors and humoral antinatriuretic mechanisms. Normalization of the hyperdynamic circulation by blockade of NO synthase (NOS) would in this context be a straightforward approach to try to prevent the sodium retention in cirrhotic liver disease, and a few studies have shown that short-term NOS inhibition in cirrhotic rats actually ameliorates the impaired sodium excretion ( 2, 35, 63 ). However, besides the circulatory effects, NO also exerts direct effects on renal tubular function, including an inhibitory action on proximal tubular sodium handling. Excess NO has been shown to inhibit the Na-K-ATPase and the type 3 sodium/proton exchanger (NHE3) in proximal tubular cell lines ( 20, 33, 46 ), and micropuncture studies in rats have shown that NO significantly decreases proximal tubular fluid reabsorption ( 14, 61 ). These micropuncture data from rats are supported by studies in humans showing that systemic administration of NOS inhibitors decreases fractional excretion of sodium (FE Na ) and fractional excretion of lithium (FE Li; an index for the delivery of tubular fluid out of the proximal tubules) ( 4, 6, 36 ). Finally, an experiment in rats has shown that a subpressor dose, i.e., a dose without effects on mean arterial pressure (MAP), of the NOS inhibitor N G - nitro- L -arginine methyl ester ( L -NAME) has significant antinatriuretic effects ( 30 ). These studies made in healthy subjects implicate a tonic inhibitory role of NO in renal sodium reabsorption. Together with studies pointing toward a beneficial effect of excess NO in cirrhosis ( 44, 54 ), the increased NOS activity in cirrhosis could implicate a protective role in counteracting the early development of sodium retention. The aim of the present study has therefore been to investigate the renal and circulatory effects of chronic blockade of the NO system in rats during the development of cirrhosis.
Rats were treated from day 1 of common bile duct ligation (CBL) with the nonselective NOS inhibitor L -NAME and followed for 5 wk, i.e., during the compensated preascitic period in which significant changes in sodium balance are initiated ( 10, 11 ). Two different doses of L -NAME were used to differentiate between effects on renal sodium handling due to direct tubular influence of NO vs. effects due to alterations in MAP. Cumulative sodium balance was followed daily for 3 wk, and after 5 wk renal plasma flow, glomerular filtration rate (GFR), and proximal tubular function were evaluated by renal clearance studies performed in fully conscious rats. Plasma levels of aldosterone and arginine vasopressin (AVP) were also measured. The functional experiments were supported by Western blotting of protein levels of renal cortical water and sodium transporters, including the electroneutral NHE3 present in the apical membrane of proximal tubules. This particular antiporter is responsible for the majority of transepithelial sodium reabsorption driven by the basolateral Na-K-ATPase ( 1, 5, 45, 57 ). The major finding of the study was that chronic NOS blockade significantly exacerbates sodium retention in rats with cirrhosis through profound effects on proximal tubular function.
METHODS
Experimental Animals
Barrier-bred and specific pathogen-free female Wistar rats (210-230 g) were obtained from Charles River (Sulzfeld, Germany). The animals were housed in a temperature (22-24°C)- and moisture (40-70%)-controlled room with a 12:12-h light-dark cycle (light on from 6 AM to 6 PM). All animals were fed a diet containing 133 mmol/kg Na, 275 mmol/kg K, and 23% protein (Altromin catalogue no. 1310, Altromin, Lage, Germany). Cirrhosis was induced during halothane-N 2 O anesthesia by CBL as described by Kountouras and co-workers ( 28 ). Briefly, biliary obstruction induces portal inflammation and bile duct proliferation, which eventually will result in the formation of cirrhosis. Control rats were operated similarly to CBL rats but without ligation of the bile duct (Sham). From the day of operation, all rats were subjected to either free access to demineralized water or L -NAME (Sigma-Aldrich Denmark, Copenhagen, Denmark) in one of two different doses given in the drinking water throughout the study period: a subpressor dose of 0.5 mg·kg body wt -1 ·day -1, previously shown to normalize NO production in cirrhotic rats ( 35, 39 ), and a 10-fold higher pressor dose, which moderately increased MAP. L -NAME concentration in the drinking water was calculated on an average weekly intake within each experimental group. The rats were followed for 5 wk, and the study was approved by national authorities and conducted in conformity with institutional guidelines that complied with national animal care laws.
Experimental Groups
Sham: no treatment
Sham-Low: sham-operated rats treated with po L -NAME (0.5 mg·kg body wt -1 ·day -1 )
Sham-High: sham-operated rats treated with po L -NAME (5.0 mg·kg body wt -1 ·day -1 )
CBL: CBL with no treatment
CBL-Low: CBL rats treated with po L -NAME (0.5 mg·kg body wt -1 ·day -1 )
CBL-High: CBL rats treated with po L -NAME (5.0 mg·kg body wt -1 ·day -1 )
Within each experimental group, the rats were divided into two subgroups and subjected to follow either of two different series of experiments, series 1 or series 2.
Series 1
Sodium balance studies (n = 5-6 in all groups). Two weeks after CBL or sham operation, the rats were transferred to metabolic cages and daily sodium balance was measured for the following 3 wk. The rats received demineralized water with or without L -NAME and granulated standard diet (Altromin catalogue no. 1310, Altromin), which contained 133 mmol Na/kg. Sodium intake was calculated from the amount of diet ingested per 24 h, and sodium loss was estimated from the amount of sodium excreted in the urine within the same period. Twenty-four-hour urine production was measured gravimetrically, and the metabolic cage was then rinsed with 40-50 ml of demineralized water to optimize the recovery of sodium. The sodium content was measured in the combined volume of urine and demineralized water, and 24-h sodium balance was then calculated as sodium intake minus urinary sodium losses (expressed per 100 g body wt). After termination of the study, the rats were anesthetized with halothane-N 2 O and the right kidney was rapidly removed, immediately frozen in liquid nitrogen, and stored at -80°C until processing for membrane fractionation.
Western blotting. The cortex from the right kidney was dissected and homogenized using a tissue homogenizer (Ultra-Turrax T8, Ika Labortechnik, Staufen, Germany) in a 3-ml ice-cold solution [300 mM sucrose, 25 mM imidazol, and 1 mM EDTA-disodium with protease inhibitors Pefablock (0.1 mg/ml) and leupeptin (4 µg/ml) and phosphatase inhibitors sodium orthovanadate (184 µg/ml), sodium fluoride (1.05 mg/ml), and okadeic acid (82 ng/ml)]. pH was adjusted to 7.2 with 0.1 M HCl, and the protein concentration in the homogenate was measured by use of a commercial kit (Pierce BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). All samples were added a dilution buffer for a final protein concentration of 3 µg/µl (486 mM Tris buffer grade, 8.7% glycerol, 104 mM SDS, 0.0875 mM bromphenol-blue, 25 mM dithiothreitol) and pH 6.8. The final sample solutions were solubilized at 60°C for 10 min. For measurements of water and sodium transporters, Western blotting was performed on cortical proteins including the water channel aquaporin-1 (AQP1) present in the proximal tubules ( 47 ), the electroneutral sodium-proton exchanger (NHE3) present in proximal and distal tubules ( 5 ), the Na-K-ATPase present in the basolateral membrane of proximal and distal tubules, the thiazide-sensitive and aldosterone-regulated ( 26 ) Na-Cl cotransporter (NCC) present in the distal convoluted tubules, and the water channel aquaporin-2 (AQP2) present in the collecting ducts ( 40 ). (For information on monoclonal and polyclonal antibodies used, please see Refs. 25, 29, and 41.) Samples were run for 90 min on 12% polyacrylamide minigels for AQP1 and AQP2 and on 7.5% polyacrylamide minigels for NHE3, Na-K-ATPase, and NCC measurements. The proteins were electrophoretically transferred from the gels to polyvinylidene difluoride membranes at 100 V for 90 min. Finally, after a 60-min 5% milk block, membranes were probed overnight at 4°C with the desired antibody. The labeling was visualized with horseradish peroxidase-conjugated secondary antibody diluted 1:3,000 (P0448; Dako, Glostrup, Denmark) using an enhanced chemiluminescence system (ECL+; Amersham, Buckinghamshire, UK) and scanned with a Fluor-S Multilmager (Bio-Rad Laboratories, Herts, UK) for quantification of individual band densitometry using the software program Quantity One (version 4.2.3, Bio-Rad Laboratories). For AQP1 and AQP2, the 29-kDa band and the 35- to 50-kDa band, corresponding to the nonglycosylated and the glycosylated protein, were scanned, for NHE3 the 87-kDa band was scanned, for Na-K-ATPase ( -1 subunit) the 96-kDa band was scanned, and for NCC the broad band centered at 165 kDa was scanned. Groups were compared as the mean of individual protein labeling from treated rats expressed relative to the labeling from a randomly assigned individual in the paired control run on the same gel.
Series 2
Renal clearance studies (n = 7-9 in all groups). Three weeks after CBL or Sham-CBL, permanent medical-grade Tygon catheters were implanted into the abdominal aorta and caval vein, and a permanent suprapubic bladder catheter was implanted into the urinary bladder as described previously ( 21, 42 ). After instrumentation, the rats were housed individually. Five weeks after CBL or Sham-CBL, the animals were transferred to restraining cages, and renal function was examined by clearance techniques in the conscious rats as previously described ( 21, 23, 42 ). The rats were adapted to the cages by being restrained for 2 h on 2 consecutive days before the final collection day to obtain unstressed conditions. Briefly, 14 tetraethylammonium bromide clearance was used as a marker for the effective renal plasma flow (ERPF), 3 inulin clearance as a marker for the GFR, and lithium clearance (C Li ) as a marker for the outflow of tubular fluid from the proximal tubules. Renal clearances (C) and fractional excretions (FE) were calculated by the standard formulas
where U x is urine concentration of x, V is urine flow rate expressed per 100 g body wt, and P x is plasma concentration of x. After a 90-min equilibration period, urine was collected for two consecutive 30-min collection periods. Arterial blood samples for plasma biochemistry were collected during the equilibration period, and arterial blood samples for clearance determination were collected before and after the urine collection periods. All blood samples were immediately replaced with heparinized blood from a normal donor rat. MAP was measured continuously throughout the entire clearance experiment. Urine volume was determined gravimetrically. Concentrations of sodium, potassium, and lithium in plasma and urine were determined by atomic absorption spectrophotometry. 3 inulin and 14 tetraethylammonium bromide in plasma and urine were determined by dual-label liquid scintillation counting.
Plasma biochemistry. The plasma concentration of aldosterone was measured by RIA using a commercial kit (Coat-A-Count Aldosterone, DPC, Los Angeles, CA). AVP was extracted from plasma in C 18 SEP-Pak cartridges and measured by RIA as described previously ( 27 ). All blood samples were taken from the arterial catheter and replaced immediately with heparinized blood from a normal donor rat.
Statistics
Comparisons between groups were performed by one-way ANOVA followed by Fisher's least significant difference post hoc test. Data from the metabolism study were displayed as cumulative changes; thus differences between groups on day 35 represented the entire period of observations. All data are presented as means ± SE, and differences were considered significant at the P < 0.05 level.
RESULTS
After 5 wk, the livers from the CBL rats had a firm consistency with a yellow, micronodular surface, along with pronounced dilatation of the proximal snip of the ligated bile duct. They all showed marked visible jaundice, and the liver weight/body weight index was markedly increased in all CBL groups ( Table 1 ). These changes were consistent with secondary biliary cirrhosis as previously described by our and other groups ( 21, 28 ).
Table 1. Body and liver weights
Series 1
Cumulative sodium balance. Figure 1 shows cumulated values for daily sodium intake ( A and B ), excretion ( C and D ), and balance ( E and F ) during the 3-wk study period ( days 15-35 after CBL or Sham-CBL). Significant differences were displayed for day 35, representing the entire period of observations. On the last day of observation, the cumulative sodium excretion was significantly lower in the CBL group compared with the sham-operated controls (CBL: 14.9 ± 0.4 mmol vs. Sham: 16.2 ± 0.3 mmol Na + /100 g body wt, excreted over 3 wk, P < 0.05). Thus because no differences between these two groups were observed in cumulative sodium intake, CBL rats had sodium retention relative to controls. Low-dose L -NAME had no effect on cumulative sodium balance in Sham rats but exacerbated sodium retention in cirrhotic rats (CBL-Low: 4.2 ± 0.4 mmol vs. CBL: 3.2 ± 0.2 mmol Na + /100 g body wt, retained over 3 wk, P < 0.05) due to the decrease in sodium excretion. High-dose L -NAME caused a significant decrease in the excretion of sodium, which caused severe sodium retention in both cirrhotic (CBL-High: 5.5 ± 0.4 mmol vs. CBL: 3.2 ± 0.2 mmol Na + /100 g body wt, retained over 3 wk, P < 0.05) and sham-operated rats (Sham-High: 3.9 ± 0.5 mmol vs. Sham: 1.9 ± 0.2 mmol Na + /100 g body wt, retained over 3 wk, P < 0.05). Cumulated sodium intake was lower in the Sham-Low group (Sham-Low: 16.6 ± 0.3 mmol vs. Sham: 18.1 ± 0.2 mmol Na + /100 g body wt, consumed over 3 wk, P < 0.05), suggesting that the spontaneous food intake was affected in this single group compared with all other groups where no differences in cumulative intake were observed.
Fig. 1. Cumulative sodium intake ( A and B ), cumulative sodium excretion ( C and D ), and cumulative sodium balance ( E and F ) calculated from 24-h measurements. SHAM, sham-operated rats; CBL, common bile duct ligation. LOW, 0.5 mg N G -nitro- L -arginine methyl ester ( L -NAME)·kg body wt (b.w.) -1 ·day -1; HIGH, 5.0 mg L -NAME·kg b.w. -1 ·day -1 (in this and subsequent figures). Values are means ± SE; n = 5-6. Significance levels are calculated on data from the last day. * P < 0.05 vs. SHAM. P < 0.05 vs. CBL.
Western blots. CORTICAL LEVELS OF AQP 1. Figure 2 A shows an example of an AQP1 Western blot of membrane fractions (15 µg protein/lane) from renal cortex. The affinity-purified anti-AQP1 protein antibody recognizes the 29- and 35- to 50-kDa band, corresponding to nonglycosylated and glycosylated AQP1 protein, respectively. Densitometry of all samples from all blots ( n = 6 in all groups) ( Fig. 2 B ) revealed that AQP1 expression was unchanged in the untreated CBL rats compared with untreated sham-operated controls (CBL: 96 ± 15% of Sham). Furthermore, L -NAME treatment had no effect on AQP1 expression in either Sham or CBL rats ( Fig. 2 C ).
Fig. 2. Western blotting of aquaporin-1 (AQP1). A : example of AQP1 labeling. B : densitomety of labeling from CBL and treated Sham groups expressed relatively to the paired Sham-control samples run on the same gel. C : densitometry of labeling from treated CBL groups expressed relative to the paired CBL control samples run on the same gel ( n = 5-6). P < 0.5 vs. control (=1.00).
CORTICAL LEVELS OF NHE 3. Figure 3 A shows an example of a Western blot of membrane fractions (15 µg protein/lane) from renal cortex. The affinity-purified anti-NHE3 protein antibody recognizes a 86-kDa band, corresponding to NHE3 protein. Densitometry of all samples from all blots ( n = 6 in all groups) ( Fig. 3 B ) revealed that NHE3 expression was significantly decreased in the untreated CBL rats compared with untreated sham-operated controls (CBL: 21 ± 7% of Sham). Furthermore, L -NAME in both subpressor and pressor doses significantly reduced the expression of NHE3 in the sham-operated rats (Sham-Low: 64 ± 11% of Sham, P < 0.05; Sham-High: 59 ± 14% of Sham, P < 0.05). Due to the weak signal in the samples from the CBL rats, we made separate Western blots ( Fig. 3 C ) with samples from the three CBL groups where the amount of protein (45 µg protein/lane) and the exposure time of the chemiluminescence signal were increased. Densitometry of these Western blots ( Fig. 3 D ) showed that NHE3 expression was unchanged in the CBL rats treated with the subpressor dose of L -NAME, whereas the pressor dose of L -NAME significantly increased the expression of NHE3 compared with untreated CBL rats (CBL-High: 375 ± 44% of CBL-control, P < 0.05). Together, these data show that L -NAME treatment reduces the expression of NHE3 in normal rats but increases the expression in CBL rats.
Fig. 3. Western blotting of type 3 Na/H exchanged (NHE3). A and C : examples of NHE3 labeling. B : densitomety of labeling from CBL and treated Sham groups expressed relative to the paired Sham-control run on the same gel. D : densitometry of labeling from treated CBL groups expressed relatively to the paired CBL control samples run on the same gel ( n = 5-6). * P < 0.05 vs. control (=1.00).
CORTICAL LEVELS OF NCC. Figure 4 A shows an example of a Western blot of membrane fractions (15 µg protein/lane) from renal cortex. The affinity-purified anti-NCC protein antibody recognizes a broad band around 161 kDa, corresponding to glycosylated NCC protein. Densitometry of all samples from all blots ( n = 6 in all groups) ( Fig. 4 B ) revealed that NCC expression was significantly decreased in the untreated CBL rats compared with untreated sham-operated controls (CBL: 54 ± 15% of Sham). L -NAME in both subpressor and pressor doses had no effect on the expression of NCC in the sham-operated rats. Due to the weak signal in the samples from the CBL rats, we made separate Western blots ( Fig. 4 C ) with samples from the three CBL groups where the amount of protein (45 µg protein/lane) and the exposure time of the chemiluminescence signal were increased. Densitometry of these Western blots ( Fig. 4 D ) showed that the NCC expression was unchanged in the CBL rats treated with both the subpressor and the pressor dose of L -NAME.
Fig. 4. Western blotting of Na-Cl cotransporter (NCC). A and C : examples of NCC labeling. B : densitomety of labeling from CBL and treated Sham groups expressed relative to the paired Sham-control samples run on the same gel. D : densitometry of labeling from treated CBL groups expressed relative to the paired CBL control samples run on the same gel ( n = 5-6). * P < 0.05 vs. control (=1.00).
CORTICAL LEVELS OF NA - K - ATPASE. Figure 5 A shows an example of a Western blot of membrane fractions (15 µg protein/lane) from renal cortex. The affinity-purified anti-Na-K-ATPase antibody recognizes a 96-kDa band, corresponding to the 1 -subunit of the Na-K-ATPase protein. Densitometry of all samples from all blots ( n = 6 in all groups) ( Fig. 5 B ) revealed that the expression was significantly decreased in the untreated CBL rats compared with untreated sham-operated controls (CBL: 47 ± 3% of Sham). Furthermore, L -NAME in both subpressor and pressor doses significantly reduced the expression in the sham-operated rats (Sham-Low: 71 ± 6% of Sham, P < 0.05; Sham-High: 52 ± 3% of Sham, P < 0.05). However, in the CBL rats ( Fig. 5 C ) the subpressor dose of L -NAME had no significant effect on Na-K-ATPase expression, whereas the pressor dose of L -NAME significantly increased the expression compared with untreated CBL rats (CBL-High: 159 ± 12% of CBL-control, P < 0.05). Together, these data show that L -NAME treatment reduces the expression of Na-K-ATPase in normal rats but increases the expression in CBL rats.
Fig. 5. Western blotting of Na-K-ATPase. A : example of Na-K-ATPase labeling. B : densitomety of labeling from CBL and treated Sham groups expressed relative to the paired Sham-control samples run on the same gel. C : densitometry of labeling from treated CBL groups expressed relative to the paired CBL control samples run on the same gel ( n = 5-6). * P < 0.05 vs. control (=1.00).
Finally, we measured cortical levels of AQP2. As previously shown ( 22 ), CBL rats had decreased levels of AQP2 in cortex compared with Sham rats (CBL: 48 ± 8% of Sham, P < 0.05, blots not shown). L -NAME had no effect on the AQP2 level in either treated Sham or treated CBL groups (data not shown).
Series 2
Plasma biochemistry. Plasma levels of aldosterone were unchanged in cirrhotic rats ( Table 2 ). L -NAME treatment had no significant effects on the plasma levels of aldosterone in cirrhotic or sham-operated rats. Plasma AVP was significantly increased in cirrhotic rats. L -NAME treatment surprisingly did not reduce the plasma levels of AVP. Plasma concentrations of sodium and potassium were similar in untreated Sham vs. CBL rats. L -NAME treatment had no effect on plasma electrolytes in the Sham groups, but the L -NAME-treated cirrhotic rats had slightly lower plasma sodium concentrations compared with the untreated CBL rats.
Table 2. Plasma biochemistry
Renal hemodynamics and GFR. MAP was not significantly decreased in the untreated CBL rats compared with the untreated Sham rats ( Table 3 ). Low-dose L -NAME treatment had no effect on MAP in either Sham or CBL rats, whereas the high-dose L -NAME treatment significantly increased MAP to 126 mmHg in both the sham-operated and cirrhotic rats. ERPF was, as previously shown ( 21 ), significantly increased in the CBL rats compared with sham-operated controls. L -NAME treatment dose dependently normalized ERPF in CBL rats, whereas L -NAME had no effect on ERPF in the sham-operated rats. GFR was significantly decreased in the CBL rats. L -NAME treatment had no effect on GFR in either Sham or CBL rats. Glomerular filtered sodium (GFR * plasma sodium concentration) was significantly lower in the CBL rats compared with Sham rats. L -NAME treatment had no effect on filtered sodium in either Sham or CBL rats.
Table 3. Renal clearance data
Renal lithium handling. C Li, but not FE Li, was significantly decreased in CBL rats, suggesting that the delivery of tubular fluid out of the proximal tubules was significantly decreased in CBL rats ( Table 3 ). Low-dose L -NAME treatment had no significant effects on renal lithium handling in either Sham or CBL rats, whereas high-dose L -NAME treatment significantly decreased both C Li and FE Li in CBL rats, suggesting that high-dose L -NAME treatment had profound effects on proximal tubular function in CBL rats. High-dose L -NAME treatment had no effect on renal lithium handling in the sham-operated rats.
DISCUSSION
The aim of the present study was to evaluate renal effects of chronic NOS inhibition during the development of cirrhosis. The level of circulating NO increases during cirrhosis, and a pathophysiological connection between this versatile gas and the development or maintenance of sodium-retaining mechanisms in cirrhosis has been intensively studied ( 3, 7, 19, 37, 38 ). Contrasting results and conclusions in the matter of beneficial vs. deleterious effects of NOS inhibition in cirrhosis have been obtained, most probably due to differences in experimental models (humans vs. animals), procedures (conscious vs. anesthetized subjects), stage of disease (preascitic vs. ascitic), choice of NOS inhibitor (specific vs. unspecific), expositional duration (acute vs. chronic), expositional doses (subpressor vs. pressor), and method of administration (systemically vs. locally). In our experimental model, untreated cirrhotic rats showed an increase in cumulative daily sodium balance compared with sham-operated rats. A significant decrease in GFR was present after 5 wk, whereas FE Li was unchanged, indicating that proximal tubular reabsorption was diminished in the CBL rats, i.e., that the absolute proximal reabsorption was reduced. Thus delivery of tubular fluid both to and from the proximal tubules was decreased in the CBL rats. Western blotting supported the finding of a reduced proximal reabsorption, showing a significant decrease in the cortical expression of NHE3 and Na-K-ATPase. It is therefore most likely that segments of the nephron other than the proximal tubule are responsible for the increased renal reabsorption in the compensated state of liver cirrhosis. Studies from both our and other groups support this finding ( 21, 48 ).
Studies using proximal tubular cell lines from different animals have shown that NO donors such as sodium nitroprusside as well as endogenous NO significantly inhibit both proximal Na-K-ATPase activity and NHE3 activity through intracellular pathways involving protein kinase C and cGMP ( 20, 32, 46, 58 ). These transporters are responsible for the majority of transepithelial sodium reabsorption in the proximal tubules ( 1, 5, 45, 57 ). Micropuncture studies in rats have shown that sodium nitroprusside added to the tubular perfusate significantly decreases proximal tubular fluid reabsorption ( 14, 61 ), and studies in humans have shown that systemic administration of NOS inhibitors have antinatriuretic effects associated with marked decreases in FE Li ( 4, 6, 36 ). These in vitro and in vivo experiments suggest that NO exerts a tonic inhibitory effect on proximal tubular sodium reabsorption.
In the present study, L -NAME treatment in a subpressor dose decreased daily sodium excretion and increased the cumulative sodium retention in CBL rats ( Fig. 1 F ). A 10-fold higher pressor dose of L -NAME induced even more severe changes in sodium accumulation also evident in the Sham rats ( Fig. 1, E and F ). The clearance studies showed that a pressor dose of L -NAME significantly decreased C Li and FE Li in CBL rats, indicating that an increased proximal tubular sodium reabsorption was responsible for the decreased daily sodium excretion. The profound effect of L -NAME treatment in CBL rats on proximal tubular function was further supported by a significant increase in the cortical expression of NHE3 and Na-K-ATPase. Together, these data show that chronic unspecific blockade of the NO system during development of cirrhosis significantly increases proximal tubular reabsorption in CBL rats. It should be outlined that a number of studies have shown that the NO system is also involved in regulating distal tubular function, including regulation of the thick ascending limb and collecting duct function, as well as NO-mediated changes in medullary blood flow that may change sodium reabsorption in medullary segments of the distal tubules ( 15 - 17, 34, 43, 53, 60 ). Even though not statistically significant, the pressor dose of L -NAME tended to increase plasma aldosterone levels in CBL rats, which could suggest an indirect effect on collecting duct sodium reabsorption. We therefore cannot exclude that changes in distal tubular function may contribute to the exacerbation in sodium retention found in the L -NAME-treated CBL rats.
The main theory connecting excess NO to sodium retention and ascites formation in cirrhosis is the peripheral arterial vasodilation theory ( 49 ). Peripheral arterial vasodilation plays a major role in the pathogenesis of the hyperdynamic circulation found in both human and experimental cirrhosis, and NO seems to play a central role as mediator ( 3, 19, 24, 37 ). It has been proposed that the development of a relative arterial underfilling (i.e., a decreased effective arterial blood volume) secondary to the arterial vasodilation activates the sympathetic nervous system, the renin-angiotensin-aldosterone system, and leads to nonosmotic release of AVP ( 49, 51 ). These antinatriuretic and antidiuretic systems could be responsible for the formation of renal sodium and water retention ( 49, 50 ), and in this context prevention of arterial vasodilation by NOS inhibition would be expected to attenuate the development of sodium retention. Indeed, it has been shown that unspecific NOS inhibition has the ability to prevent arterial vasodilation in a model of portal hypertension ( 31 ) and that nonspecific or specific neuronal NOS inhibition for 1 wk attenuates renal sodium and water excretion in rats with severe decompensated liver cirrhosis induced by carbon tetrachloride ( 35, 63 ). Thus Martin and co-workers ( 35 ) showed that the same subpressor dose of L -NAME as used in the present study significantly increased renal sodium excretion. A possible explanation for these results, which conflict with the present findings, could be that increased plasma levels of renin and aldosterone observed at the decompensated state of cirrhosis were normalized by NOS inhibition ( 35, 39 ). In our study, the dose-dependent reduction in renal blood flow found in CBL rats during L -NAME treatment could be interpreted as a normalization of the arterial vasodilation caused by excess NO. L -NAME treatment did not affect the circulating levels of aldosterone or vasopressin, and potential effects of L -NAME on circulating levels of aldosterone therefore did not affect overall tubular sodium excretion in our study.
Our study design, with a primary focus on proximal tubular function, limits definite conclusions on the interaction between the NO system and overall tubular (full-length nephron) sodium and water handling. It has been shown in a wide number of both in vitro and in vivo settings that NO influences distal sodium and water transport (e.g., tubular fluid, medullary osmotic gradients, or medullary blood flow). We have limited our focus on distal function to include plasma levels of AVP and aldosterone (and the protein levels of NCC), and the results allow us to conclude that if overall tubular sodium retention is dependent on the distal part of the nephron, it is most likely not mediated through aldosterone-mediated mechanisms. Furthermore, it is important to recognize Western blotting as a method of relative quantification of proteins between groups. All results are quantitative measures of cortical proteins, and it is not specified whether these proteins actually arise from the proximal tubules or other tubular segments present in the cortex. Although both Na-K-ATPase and NHE3 are expressed in the thick ascending limb of Henle, both functionality and activity of NHE3 in this segment are questioned ( 1, 5, 57 ). Although our findings are limited in the specificity of focal L -NAME effects, the overall results make it possible to hypothesize that an increase in endogenous NO plays a protective role in the maintenance of proximal tubular function, counteracting pathophysiological activation of sodium-retaining mechanisms such as renal sympathetic nerve activity ( 12 ) or intrarenal ANG II ( 18, 64 ) in preascitic cirrhosis.
DiBona and co-workers ( 10, 12, 13 ) have demonstrated that the development of sodium retention in CBL rats is dependent on the activation of renal sympathetic nerve activity, and other studies point toward beneficial effects on renal sodium handling with low-dose losartan treatment in CBL rats ( 64 ). The renal sympathetic nerves are important modulators of renal sodium excretion through release of the neurotransmitter norepinephrine ( 8 ), and ANG II enhances proximal reabsorption through activation of Na-K-ATPase expression ( 59 ). Furthermore, a mutual intrarenal dependence between ANG II and renal sympathetic activity has been described ( 9 ), and recent studies point toward a significant role of NO in modulating both of the systems. Thus ANG II stimulates neuronal NOS and renal interstitial cGMP production, probably via the AT 2 receptor ( 52 ), and recent work by Zhang and Mayeux ( 65 ) shows that neuronal NOS-derived NO is triggered beyond a certain ANG II threshold concentration, thereby counteracting the stimulatory effect on the Na-K-ATPase. Similarly, studies have shown that NO exerts a tonic inhibitory action on nerve-mediated proximal tubular reabsorption, and at the same time NO exerts a facilitatory role in norepinephrine release ( 55, 61, 62 ). Altogether these experiments show that NO is an integrated modulating agent in renal proximal tubular function, further studies on the interaction between the NO system and other modulators of proximal tubular sodium handling in cirrhosis are warranted.
In summary, chronic L -NAME treatment exacerbates renal sodium retention in rats with cirrhosis induced by CBL. Increased proximal tubular sodium reabsorption is the main mechanism involved, probably mediated through an increased expression of apical NHE3 and basolateral Na-K-ATPase. The effect of NOS inhibition found in CBL rats indicates a pivotal role for NO in counteracting proximal tubular dysfunction in cirrhosis, and we therefore suggest that chronic L -NAME treatment unmasks the effect of intensive sodium-retaining mechanisms otherwise suppressed by increased NO. These mechanisms could include the intrarenal actions of ANG II, renal sympathetic nerve activity, or other neurohumoral agents with influence on renal sodium handling in preascitic cirrhosis.
GRANTS
This work received financial support from The Danish Medical Research Council, the Danish Heart Foundation, The Novo Nordic Foundation, The Eva and Robert Voss Hansen Foundation, The Ruth Kønig-Petersen Foundation, The Helen and Ejnar Bjørnow Foundation, and the European Commission (QRLT 2000 00778 and QRLT 2000 00987).
ACKNOWLEDGMENTS
The technical assistance of Anette Nielsen, Iben Nielsen, Bettina Sandborg, Barbara Seider, Haya Holmegaard, Louise Frandsen, and Helle Høyer is acknowledged. We gratefully acknowledge Dr. J. Warberg for performing the plasma AVP analyses.
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作者单位：1 Department of Pharmacology, University of Copenhagen, DK-2200 Copenhagen N; 2 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C; and 3 Department of Neuroanesthesia, The Neuroscience Center, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmark