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Am J Physiol Gastrointest Liver Physiol 294: G567-G575, 2008. First published January 3, 2008; doi:10.1152/ajpgi.00232.2007
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LIVER AND BILIARY TRACT

LPS induces the TNF-{alpha}-mediated downregulation of rat liver aquaporin-8: role in sepsis-associated cholestasis

Guillermo L. Lehmann, Flavia I. Carreras, Leandro R. Soria, Sergio A. Gradilone, and Raúl A. Marinelli

Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina

Submitted 22 May 2007 ; accepted in final form 2 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although bacterial lipopolysaccharides (LPS) are known to cause cholestasis in sepsis, the molecular mechanisms accounting for this effect are only partially known. Because aquaporin-8 (AQP8) seems to facilitate the canalicular osmotic water movement during hepatocyte bile formation, we studied its gene and functional expression in LPS-induced cholestasis. By subcellular fractionation and immunoblotting analysis, we found that 34-kDa AQP8 was significantly decreased by 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, expression and subcellular localization of hepatocyte sinusoidal AQP9 were unaffected. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (Pf) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (65 ± 1 vs. 49 ± 1 µm/s) by LPS, consistent with defective canalicular AQP8 functional expression. By Northern blot analysis, we found that 1.5-kb AQP8 mRNA expression was increased by 80%, suggesting a posttranscriptional mechanism of protein reduction. The tumor necrosis factor-{alpha} (TNF-{alpha}) receptor fusion protein TNFp75:Fc prevented the LPS-induced impairment of AQP8 expression and bile flow, suggesting the cytokine TNF-{alpha} as a major mediator of LPS effect. Accordingly, studies in hepatocyte primary cultures indicated that recombinant TNF-{alpha} downregulated AQP8. The effect of TNF-{alpha} was prevented by the lysosomal protease inhibitors leupeptin or chloroquine or by the proteasome inhibitors MG132 or lactacystin, suggesting a cytokine-induced AQP8 proteolysis. In conclusion, our data suggest that LPS induces the TNF-{alpha}-mediated posttranscriptional downregulation of AQP8 functional expression in hepatocytes, a mechanism potentially relevant to the molecular pathogenesis of sepsis-associated cholestasis.

bile secretion; water channels; hepatocyte; water transport

AQUAPORINS (AQPs) are a family of homologous tetrameric proteins that facilitates the osmotically driven water transport through cell membranes (24). At least 13 aquaporin isoforms have been identified from various mammalian tissues, of which four are expressed in rat hepatocytes: AQP8 (4, 9, 12, 22), AQP9 (10, 22, 45), AQP11 (16), and AQP0 (22). Among them, AQP8 has been largely studied in hepatocytes, where it is localized as a glycosylated 34-kDa protein in the canalicular plasma membrane domain (4, 9, 12, 22) and within the cell in vesicular compartments (4, 9, 12, 22) and as a nonglycosylated 28-kDa protein in mitochondria (3). Under specific stimulus such as the hormone glucagon (17, 18, 36) or its second messenger cAMP (12, 22), intracellular AQP8 redistributes to the hepatocyte canalicular domain, increasing the canalicular osmotic water permeability and thus facilitating the osmotic movement of water.

Canalicular bile secretion is a coordinated process resulting from the interaction between solute membrane transport and the rapid movement of water into the biliary space in response to the transient osmotic gradient generated (1). There is experimental evidence suggesting that AQP8 facilitates the osmotically driven canalicular membrane water transport (34) and bile formation in rat hepatocytes (22). In line with this, recent studies reported that AQP8 is downregulated in animal models of obstructive and estrogen-induced cholestasis (5, 6).

It is well known that sepsis is commonly accompanied by cholestasis (15). Lipopolysaccharides (LPS) are endotoxins released into circulation from bacterial sites of infection, which are responsible for the macrophage (i.e., Kupffer cells) secretion of proinflammatory cytokines, primarily tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL) 1-β, and IL-6. These cytokines are thought to be the principal mediators of bile secretory failure (15).

LPS administration to rodents represents an established experimental model of sepsis that is being associated with significant changes in gene expression and functioning of several hepatic membrane transporters implicated in the development of bile secretory failure. Therefore, LPS provides a helpful model to clarify mechanistic events in sepsis-induced cholestasis. As the precise molecular mechanisms for LPS-induced cholestasis are incompletely defined, our aim was to investigate the involvement of hepatocyte AQPs in this disorder.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model for LPS-induced cholestasis. Adult male Wistar rats were maintained on a standard laboratory diet and water ad libitum and housed in a temperature- and humidity-controlled environment under a constant 12-h light-dark cycle. All animals received humane care, according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Protocols were approved by the local animal welfare committee. The experimental model was induced by injecting rats via the femoral vein with Salmonella typhimurium LPS (4 mg/kg body wt) (Sigma, St. Louis, MO) dissolved in sterile 0.9% NaCl under ether anesthesia. Control rats were injected with saline only. LPS increased the plasma levels of cytokines TNF-{alpha} (49 vs. 1,765 pg/ml, 2 h after administration), IL-1β (139 vs. 590 pg/ml, 4 h after administration), and IL-6 (550 vs. 25,187 pg/ml, 4 h after administration). After 16 h, animals were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and bile was collected by 30 min. Bile flow was determined by gravimetry (6). Animals were euthanized, and livers were harvested for evaluation. LPS caused cholestasis as indicated by a 30% decrease in bile flow compared with control rats (i.e., 6.3 ± 0.5 vs. 4.4 ± 0.3 µl·min–1·100 g–1 body wt, P < 0.05).

Inactivation of TNF-{alpha} in plasma. TNF-{alpha} blockade was performed by intraperitoneal injections of a soluble p75 TNF-{alpha} receptor fusion protein (TNFp75:Fc) (Amgen, Thousand Oaks, CA) at a dose of 8 mg/kg body wt, 16 h and 1 h before the LPS injection (14). Controls received saline alone. TNFp75:Fc prevented the LPS-induced increase of TNF-{alpha} plasma levels (1,765 vs. 220 pg/ml) without blocking those of cytokines IL-1β (590 vs. 714 pg/ml) and IL-6 (25,187 vs. 42,600 pg/ml).

Isolation, culture, and treatment of hepatocytes. Hepatocytes isolated from livers of male Wistar rats by collagenase perfusion and mechanical disruption (12) were cultured as described (6) in the presence or absence of recombinant human TNF-{alpha} (1,000 U/ml) (Promega, Madison, WI) for 8 h. Under these conditions, TNF-{alpha} decreased hepatocyte AQP8 protein expression. Some groups were preincubated for 30 min in the presence of the lysosomal inhibitors leupeptin (250 µM) (Chemicon, Temecula, CA) (6) or chloroquine (100 µM) (Sigma) (29), or the proteasomal inhibitors MG132 (10 µM) (Sigma) (6) and lactacystin (5 µM) (29, 32) (Calbiochem, San Diego, CA). Total hepatocyte membranes were prepared as described (6).

Cell viability. The effect of TNF-{alpha} on cell viability was assessed by a standard Trypan blue exclusion assay and by a lactate dehydrogenase-release assay (42). Cell cultures were also examined morphologically by light microscopy.

Preparation of hepatic subcellular membrane fractions. Livers were homogenized by 15 up-and-down strokes with a loose-fitting Dounce homogenizer in four volumes of 0.3 M sucrose containing 0.1 mM phenylmethylsulfonyl fluoride and 0.1 mM leupeptin (Sigma). Liver homogenates (or sonicated cultured hepatocytes) were subjected to low-speed centrifugation to obtain postnuclear supernatants and then centrifuged at 200,000 g for 60 min, yielding the total liver or hepatocyte membrane fractions (12). Fractions enriched in plasma or intracellular microsomal membranes were prepared from liver homogenates by centrifugation at 200,000 g for 60 min on a discontinuous 1.3 M sucrose gradient as previously described (5). The plasma membrane band was removed, diluted to 0.3 M, and centrifuged at 200,000 g for 60 min to yield the plasma membrane fraction. The gradient remainder was sonicated, diluted to 0.3 M, and centrifuged at 17,000 g for 30 min. The resulting supernatant was centrifuged at 200,000 g for 60 min to yield the intracellular microsomal membrane fraction. The plasma membrane fraction was further purified by centrifugation at 100,000 g for 90 min on a continuous (9–60%) sucrose gradient. Membrane proteins were determined by Lowry et al. (30). The purity of plasma and intracellular membranes was assessed by enzyme markers 5'-nucleotidase, acid phosphatase, and aspartate aminotransferase and was assessed with commercial kits (Wiener Lab, Rosario, Argentina). Glucose-6-phosphatase was determined as described (5). The data obtained are summarized in Table 1. The enrichment and purity of membrane fractions were comparable to those reported previously (5, 6) and similar between LPS and control rats.


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  		Table 1. Assessment of plasma and microsomal membrane purity

 
Preparation of canalicular liver plasma membranes. Canalicular plasma membranes were prepared from livers as previously described by our laboratory (6, 34). Briefly, purified plasma membrane fractions obtained as described above (see purity on Table 1) were layered onto a discontinuous sucrose gradient composed of 31, 34, and 38% sucrose. After centrifugation at 270,000 g for 3 h, the band at the top of the 31% layer (canalicular plasma membrane) was collected, diluted with five volumes of washing buffer, and spun at 200,000 g for 1 h. Livers from control and LPS-treated rats were processed in parallel. The purity of the canalicular plasma membrane fraction was assessed by the canalicular enzyme marker 5'-nucleotidase as mentioned above and the basolateral marker Na+/K+ ATPase according to Scharschmidt et al. (39). The data obtained are summarized in Table 2. The enrichment and purity of the canalicular membranes were comparable to those reported previously (22) and similar between LPS and control rats. Total cholesterol from canalicular fractions was measured by the cholesterol oxidase method (38).


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  		Table 2. Assessment of canalicular plasma membrane purity

 
Vesicle size measurements. To determine the size of vesicles, electron micrographs were taken on a ME 10 Zeiss electron microscope. Pellets of membrane vesicles were fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.2), treated with 1% OsO4, washed with the same buffer, and then treated with uranyl acetate. After dehydration and embedding in the resin Durcupan, thin sections were stained with lead citrate. Size measurements were made on micrographs at x30,000 magnification with the software Image-Pro Plus (Media Cybernetics, Silver Spring, MD). The software was set to select objects (i.e., vesicular structures) in the range of 50–500 nm (6, 34).

Stopped-flow light scattering. The time course of vesicular volume was followed from changes in scattered light intensity at 450 nm by using a SX.18MVR stopped-flow spectrometer (Applied Photophysics, Surrey, UK), which has a 1.3-ms dead time and 99% mixing efficiency in <1 ms. Experiments were performed at 20°C; 50 µl of a concentrated vesicle suspension were diluted into 450 µl of 50 mM sucrose and 5 mM HEPES-Tris, pH 7.4. One of the syringes of the stopped-flow apparatus was filled with the membrane suspension, whereas the other was filled with the same buffer containing sucrose to establish a hypertonic gradient of 250 mosM upon mixing. The final protein concentration after mixing was 150 µg/ml. Immediately after applying a hypertonic gradient, water outflow occurs and the vesicles shrink, causing an increase in scattered light intensity. The data were fitted to single exponential functions. The osmotic water permeability coefficient (Pf) was calculated as described (6, 34), using the equation: Pf = Kexp·Vo/Av·Vw·{Delta}C, where Kexp is the fitted exponential rate constant, V0 is the initial mean vesicle volume, Av is the mean vesicle surface, Vw is the molar volume of water, and {Delta}C is the osmotic gradient. V0 and Av were calculated by using a mean canalicular vesicle diameter assessed as described above.

Serum cytokine measurements. Serum concentrations of rat TNF-{alpha}, IL-1β, and IL-6 were measured by the ELISA method according to the protocol provided by the manufacturer (eBioscience, San Diego, CA for TNF-{alpha} and IL-1β; Biosource International, Camarillo, CA for IL-6).

Immunoblotting. Membrane fractions were subjected to 12% SDS-PAGE and transferred to polyvinyl difluoride membranes. After being blocked and washed, blots were incubated overnight at 4°C with rabbit affinity-purified antibodies against AQP8 or AQP9 as described (6) or mouse antibody against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Densitometry was performed using Gel-Pro3 software (Media Cybernetics).

Immunohistochemistry. Livers were fixed, processed, and embedded in paraffin as described (5). AQP8 and AQP9 were localized with affinity-purified antibodies (10 µg/ml; Alpha Diagnostics International, San Antonio, TX) and horseradish peroxidase-conjugated secondary antibody (DakoCytomation, Glostrup, Denmark) on serial 5-µm sections. Controls were performed by omitting the primary antibodies.

Northern blot analysis. Total RNA from livers was processed and hybridized with a specific rat AQP8 complementary DNA probe labeled with {alpha}-32P deoxycytidine 5'-triphosphate, as described (6).

Statistical analysis. Data are means ± SE Statistical significance was determined by Student's t-test or one-way ANOVA, Tukey's test; P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of AQP proteins in LPS-induced cholestasis. To examine the AQP protein expression and subcellular localization in LPS-induced cholestasis, we performed immunoblotting on liver membrane fractions. As seen on Fig. 1A, AQP8 protein levels were significantly reduced by 55% in plasma membranes and 84% in intracellular membranes. Accordingly, AQP8 in total membranes was reduced by about 60%. LPS treatment did not significantly affect AQP9 protein levels in total or plasma membranes (Fig. 1B). Consistent with the predominant hepatocyte surface localization of AQP9 (22), intracellular membranes showed no detectable signal.


Figure 1
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  		Fig. 1. Immunoblotting for aquaporin-8 (AQP8) and AQP9 in LPS-induced cholestatic liver. Rats were injected with LPS (4 mg/kg body wt) or saline alone. After 16 h, livers were collected and processed (see MATERIALS AND METHODS). A: representative immunoblottings for AQP8 in total (TM), plasma (PM), and intracellular microsomal membranes (IM) (30 µg protein/lane) with corresponding densitometric analysis (n = 4). The blots were reprobed by using anti-β-actin antibody as a control for equal protein loading. B: representative immunoblottings for AQP9 in TM and PM with corresponding densitometric analysis (n = 4). The blots were reprobed by using anti-β-actin antibody as a control for equal protein loading. AQP9 was undetected in IM. Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
Immunohistochemical staining for both AQPs in serial liver sections was performed to confirm the effect of LPS on the expression and subcellular localization of hepatocyte AQP8 and AQP9 (Fig. 2). Immunohistochemical data were in agreement with immunoblot analysis. As observed previously (4–6, 22), AQP8 in control rat livers was found to be predominantly intracellular in hepatocytes, although immunoreactivity was also observed on the canalicular plasma membrane (Fig. 2A). After 16 h of LPS administration, AQP8 staining was homogeneously decreased (Fig. 2B). The staining pattern for AQP9 in control livers was mainly observed on the hepatocyte sinusoidal plasma membranes (Fig. 2D) in agreement with previous reports (6, 22). AQP9 staining remained unaltered in LPS-treated livers (Fig. 2E). Similar immunostaining patterns were observed in three separate control and LPS-treated rats. Immunohistochemical controls showed no labeling (Fig. 2, C and F).


Figure 2
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  		Fig. 2. Immunohistochemistry for AQP8 and AQP9 in LPS-induced cholestatic liver. A: AQP8 in control rats. Immunoreactivity (brown staining) was seen on the canalicular plasma membrane (arrow, inset) as well as in intracellular membrane compartments of hepatocytes. B: AQP8 in cholestatic rats, where the labeling was uniformly decreased. D: AQP9 in control rats. Immunoreactivity (brown staining) was mainly seen on the sinusoidal plasma membrane of hepatocytes. E: AQP9 in LPS-induced cholestasis. Labeling was not significantly changed. C and F: no staining was observed in experiments omitting the AQP8 or AQP9 primary antibodies. Original magnification x400.

 
Water permeability of canalicular membranes from LPS-induced cholestatic liver. To study whether LPS-induced AQP8 downregulation caused a reduction in canalicular water permeability, we measured the osmotic water permeability by stopped-flow spectrophotometry. Fig. 3A shows typical tracings of a time course of scattered light intensity (water transport) in canalicular plasma membrane vesicles from normal and LPS-cholestatic livers in response to a 250 mosM hypertonic sucrose gradient. No change in scattered light was observed when vesicles were mixed with isosmotic buffer, showing absence of mixing artifacts. Data fit well to a single exponential function, indicating the presence of functional homogenous populations of canalicular vesicles. The corresponding calculated Pf values are shown in Fig. 3B. The canalicular Pf value for control rats was 65 ± 1 µm/s, comparable to that reported previously (6, 34). Canalicular Pf for LPS-cholestatic rats was significantly reduced (49 ± 1 µm/s; P < 0.05). To further assess the quality of the canalicular membranes used in these studies, we performed immunoblotting for AQP8 and for hepatocyte sinusoidal AQP9 (Fig. 3C). As expected, the canalicular membranes showed an enrichment in AQP8 with only negligible amounts of AQP9 in control as well as in LPS-treated rats. These data, together with those of Table 2, are indicative of the purity and minor degree of contamination of the canalicular by the basolateral membranes. Figure 3D shows, consistently with data from Figs. 1 and 2, a significant reduction of about 70% in the canalicular AQP8 protein level after LPS treatment.


Figure 3
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  		Fig. 3. Water permeability of canalicular membranes from LPS-induced cholestatic liver. Rats were injected with LPS (4 mg/kg body wt) or saline alone. After 16 h, livers were collected and processed (see MATERIALS AND METHODS). A: typical tracings of a time course of scattered light intensity (osmotic water transport), along with single exponential fits, in canalicular plasma membrane vesicles from normal and cholestatic livers in response to a 250 mosM hypertonic sucrose gradient. No change in scattered light was observed when vesicles were mixed with isosmotic buffer (isosmolar). B: osmotic water permeability (Pf) values calculated from the rate constant of the single-exponential fits, as described in MATERIALS AND METHODS. Data are means ± SE from 3 independent vesicle preparations. *P < 0.05. C: AQP expression in the canalicular plasma membrane fraction. Representative immunoblottings for AQP8 and AQP9 in liver homogenate and canalicular plasma membrane (15 µg protein/lane) from control and LPS-treated rats. D: anti-AQP8 immunoblot of the canalicular plasma membranes used for Pf assessment (15 µg protein/lane) with corresponding densitometric analysis (n = 3). The blot was reprobed by using anti-β-actin antibody as a control for equal protein loading. Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
Mean canalicular vesicle diameter from control (175 ± 4 nm; n = 1,080) and LPS-treated livers (171 ± 5 nm; n = 1,050) was not significantly different. Thus the decreased canalicular Pf in LPS-treated rats cannot be due to a difference in either the initial vesicle size or the purification of the membranes (see MATERIALS AND METHODS).

Steady-state AQP8 mRNA levels in LPS-induced cholestasis. To begin to explore the mechanisms of LPS-induced AQP8 protein downregulation, we assessed the steady-state mRNA levels by Northern blot analysis. AQP8 mRNA expression was found not to be reduced but actually increased by 80% with the treatment (Fig. 4). The increased level of mRNA, together with the reduced AQP8 protein expression, suggests the involvement of posttranscriptional mechanisms.


Figure 4
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  		Fig. 4. Northern blot analysis for AQP8 mRNA in LPS-induced cholestatic liver. Total RNA was isolated and processed from livers of control and LPS-treated rats. A: autoradiographs for AQP8 mRNA corresponding to 3 independent experiments per group. The ethidium bromide staining of the gel indicating the presence of 28S ribosomal RNA (rRNA) is shown to confirm mRNA integrity and equal loading. B: densitometric analysis of AQP8 steady-state mRNA levels (n = 3). Expression of the AQP8 mRNA was normalized against the expression of the 28S rRNA, which was not altered with the treatment. Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
Effect of TNF-{alpha} inactivation on AQP8 protein expression in LPS-induced cholestasis. To determine if the reduced AQP8 protein expression in LPS-induced cholestasis was TNF-{alpha}-mediated, we neutralized in vivo the plasma TNF-{alpha} levels, making use of the fusion protein TNFp75:Fc. As shown in Fig. 5, LPS administration reduced the AQP8 protein expression in both total and plasma membranes (56 and 58%, respectively). TNFp75:Fc completely prevented the LPS-induced effect, suggesting a central role for TNF-{alpha} in the LPS-induced AQP8 downregulation. Accordingly, the LPS-induced bile flow reduction was also prevented by TNFp75:Fc.


Figure 5
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  		Fig. 5. Effect of the in vivo TNF-{alpha} inactivation on LPS-induced AQP8 downregulation and bile flow. TNF-{alpha} blockade was performed by administration of TNFp75:Fc (8 mg/kg body wt) as in MATERIALS AND METHODS. A: representative immunoblottings for AQP8 in TM and PM fractions (30 µg protein/lane). The blots were reprobed by using anti-β-actin antibody as a control for equal protein loading. B: corresponding densitometric analysis (n = 4). C: bile flow. Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
Role of lysosomal and proteasomal proteolytic pathways in TNF-{alpha}-induced AQP8 downregulation. An in vitro model was established to confirm the effects of TNF-{alpha} on hepatocyte AQP8 protein expression and to investigate the effects of lysosomal and proteasomal inactivation in the AQP8 protein downregulation. We had previously observed that AQP expression in primary hepatocyte cultures remains stable up to 24 h (6). Examination by light microscopy demonstrated that cells treated with TNF-{alpha} remained adherent and morphologically similar to control cells. Treated hepatocytes exhibited no significant differences in cell viability as determined by lactate dehydrogenase-release assay. Consistent with this, the number of live cells (Trypan blue exclusion) and the protein content after 8 h of exposure to TNF-{alpha} were equal in control and treated cells.

In agreement with our in vivo observations (Fig. 5), TNF-{alpha} decreased AQP8 protein expression in cultured hepatocytes by about 45%, whereas that of AQP9 remained unchanged (Fig. 6A).


Figure 6
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  		Fig. 6. Effect of TNF-{alpha} and protein-processing inhibitors on AQP expression in cultured hepatocytes. Primary hepatocytes were exposed 8 h to TNF-{alpha} (1,000 U/ml). A: representative immunoblottings for AQP8 and AQP9 in TM fractions. B: cell cultures were preincubated for 30 min in the presence of lysosomal protease inhibitors leupeptin (250 µM) or chloroquine (100 µM). C: the proteasome inhibitors MG132 (10 µM) or lactacystin (5 µM) and then for additional 8 h in the absence (–) or presence (+) of TNF-{alpha}. Anti-AQP8 and AQP9 immunoblots of total hepatocyte membranes (20 µg protein/lane) and corresponding densitometric analysis are shown (n = 6). The blots were reprobed by using anti-β-actin antibody as a control for equal protein loading. Data (means ± SE) are expressed as percentage of controls. *P < 0.05.

 
Pretreatment with the lysosomal protease inhibitors leupeptin or chloroquine prevented the TNF-{alpha}-induced AQP8 downregulation (Fig. 6B). The proteasomal inhibitors MG132 and lactacystin were also able to prevent the AQP8 protein downregulation (Fig. 6C). These results indicate that TNF-{alpha} induced the degradation of hepatocyte AQP8 via both the lysosomal and proteasomal proteolytic pathways.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrated for the first time that LPS reduced the functional expression of hepatocyte canalicular AQP8 through posttranscriptional mechanisms mediated by TNF-{alpha}. This finding supports a contributing role of AQP8 to LPS-induced bile secretory dysfunction.

Cholestasis in sepsis is thought to result mainly from a functional defect in bile formation at hepatocyte level. LPS, released into the circulation, induces the secretion of proinflammatory cytokines, mainly IL-1β, IL-6, and TNF-{alpha}, from activated Kupffer cells. Hepatocytes respond to these cytokines with a reduction in gene expression of solute transport proteins, which, in turn, would lead to bile secretory failure (15).

Bile secretion is an osmotic secretory process resulting from the inflow of water into the biliary space in response to osmotic gradients created mainly by the excretion of bile salts via the bile salt transporter Bsep, glutathione via the organic anion transporter Mrp2, and HCO3– via the Cl–/HCO3– exchanger AE2 (1). The resulting water flow occurs either transcellularly or through the tight junctions. Although the quantitative significance of these two pathways of water flow is not yet fully established, there is compelling evidence supporting a primary role for the transcellular pathway (2, 22, 33–35). Water permeability in hepatocytes is partially explained by the existence of AQPs. Whereas AQP8 modulates the canalicular, rate-limiting water flow, AQP9 contributes to the sinusoidal uptake (22, 34). The development of AQP-null mice has been useful in providing insight into the role of AQPs in epithelial water transport. A recent study using AQP8-knockout mice found no significant dietary fat misprocessing (47). Although this finding suggests a preserved excretion of bile salts for lipid digestion, direct studies on bile formation to assess whether AQP8-knockout mice develop cholestasis are still pending.

It has been clearly documented that, as shown in this study, rat hepatocyte AQP8 is localized in intracellular vesicular compartments as well as on canalicular, but not basolateral, plasma membranes (4, 5, 9, 12, 22, 41). Nevertheless, there are conflicting data about the subcellular localization of mouse hepatocyte AQP8. Immunohistochemical and immunogold electron microscopy studies showed similar AQP8 localization (i.e., in intracellular compartments and on canalicular membrane) between mouse and rat hepatocytes (11). However, a immunofluorescence study showed a predominant plasma membrane (basolateral) localization of liver mouse AQP8 (47).

Bile flow therefore results from the functional and molecular expression of canalicular transport systems, combined with an adequate canalicular water permeability determined by both AQPs and lipid composition. Hence, it can be hypothesized that altered AQP8 plasma membrane expression is associated with an altered bile physiology. In line with this, cholestasis by bile duct ligation shows impaired trafficking and expression of AQP8 (5). In addition, intrahepatic cholestasis induced by either estrogens (6) or LPS, as shown here, displays substantial downregulation of hepatocyte AQP8 together with an impaired canalicular osmotic water transport. Thus extrahepatic and hepatocellular cholestasis are associated with altered hepatocyte plasma membrane AQP8 expression. As reported here for AQP8, LPS-treated rodents show protein downregulation of the canalicular export proteins, Bsep and Mrp2 (28). Both transporters are gene regulated in response to LPS, in combination with posttranscriptional events in the early onset of LPS-induced cholestasis such as the rapid retrieval of Mrp2 from the canalicular membrane (25). Although the HCO3– excretion into bile was shown to be impaired in endotoxemic rats, the functional and molecular basis of this phenomenon remains to be clarified (44). In contrast to the lack of effect of LPS treatment shown here on basolateral AQP9, the expression of some solute basolateral transporters, e.g., Na+-taurocholate-cotransporting polypeptide (NTCP) (19), is significantly reduced. Furthermore, NTCP experiences rapid retrieval from the basolateral membrane after LPS stimulation (26).

Water transport across cell membranes occurs through the lipid bilayer or across water channels in response to osmotic gradients. Previous studies have demonstrated the coexistence of lipid- and AQP-based routes for water movement across hepatocyte canalicular plasma membranes. Under unstimulated conditions, it has been roughly estimated that AQP water-mediated pathway contributes to about 30% of the overall canalicular water transport (34). Accordingly, our findings show a decrease of ~70% in canalicular AQP8 protein expression, in correspondence with a 25% decreased water canalicular permeability, measured by stopped-flow spectrophotometry (Fig. 3). This result is in good agreement with previous studies in estrogen-induced cholestasis (6). The water permeability of the lipid-mediated pathway is known to be disturbed with increased cholesterol level (7). Nevertheless, cholesterol content in our canalicular membranes was not significantly different between control and LPS-treated groups (0.26 ± 0.09 vs. 0.20 ± 0.05 µmol/mg protein), which strongly suggests a decrease in the fraction of water moving through the AQP pathway instead of across the lipid bilayer. Thus LPS-induced cholestasis may ultimately be caused by an impairment of the transient osmotic gradients generated by defective canalicular expression of the solute transporters Bsep and Mrp2, together with reduced canalicular water permeability secondary to defective AQP8 expression.

As above stated, TNF-{alpha} and other proinflammatory cytokines are putative mediators in sepsis-induced cholestasis. Accordingly, passive immunization with anti-TNF-{alpha} antibody prevented LPS-induced cholestasis in vivo (46). Studies in mice reveal that TNF-{alpha} and IL-1β downregulate the gene expression of Mrp2, Bsep, Oatp1, and NTCP (13). Furthermore, cultured rat hepatocytes exposed to TNF-{alpha} exhibit impaired bile salt uptake (46). Our in vivo TNF-{alpha} inactivation studies show a complete prevention of AQP8 downregulation and bile flow reduction in the LPS-treated rats. We confirmed the TNF-{alpha} effects on AQP8 in hepatocyte primary cultures, an in vitro model that excludes other potential LPS and TNF-{alpha} in vivo effects, such as circulatory alterations or release of other cytokines from Kupffer cells.

We found that steady-state AQP8 mRNA levels are increased in LPS-induced cholestasis. The observed downregulation of AQP8 protein, together with an increased AQP8 mRNA, suggests a common compensatory mechanism in cholestasis, as reported previously by our laboratory in obstructive (5) and estrogen-induced cholestasis (6). Moreover, in accordance with our previous observations (5, 6), AQP8 downregulation does not seem to be related to any major modification of the AQP8 transcript (see Fig. 4) or AQP8 molecular mass (34 kDa), which indicates proper protein glycosylation. Regarding the precise mechanisms involved in the LPS-induced AQP8 protein downregulation, our data suggest to be posttranscriptional. Interestingly, although rodent canalicular solute transporters seem to be regulated transcriptionally in response to LPS (28), recent evidence obtained from human liver slices suggests that Mrp2 and Bsep regulation is mainly posttranscriptional (8).

In the present study we show that both lysosomal and proteasomal pathways participate in the protein degradation of AQP8 in TNF-{alpha}-treated cultured rat hepatocytes. Indeed, by using the lysosomal protease inhibitors leupeptin and chloroquine, or the proteasome inhibitors lactacystin and MG132, we could consistently prevent the TNF-{alpha}-induced AQP8 downregulation. Other AQP proteins not expressed in hepatocytes (i.e., AQPs 1, 2, 4, and 5), are targeted for proteolysis through the lysosomal or the proteasome system (20, 29, 31, 40). Moreover, we have previously shown that the lysosome pathway is involved in the estrogen-induced AQP8 downregulation (6). To our knowledge, this is the first study reporting the TNF-{alpha}-mediated proteasomal degradation of an AQP water channel. A recent study in murine lung epithelial cells reported that TNF-{alpha} downregulated AQP5 both pre- and posttranslationally, although degradation pathways were not investigated (43). The degradation of numerous cytoplasmic (32) and membrane proteins, such as AQP2 (20, 21), platelet-derived-growth factor receptor (37), Met tyrosine kinase receptor (23), and the gap junction subunit Cx43 (27) appears to be prevented by inhibitors of both lysosomal and proteasomal degradation systems. Nevertheless, the functional association between these degradative pathways remains obscure. A previous study (20) performed in mpkCCDc14 cells suggested that the effective degradation of AQP2 by one of the proteolytic pathways requires the previous degradation of an additional protein by the other pathway. This suggestion may be valid in our experimental model, as the inhibition of either route was able to prevent the TNF-induced AQP8 downregulation. Further studies will be necessary to clarify this issue.

In conclusion, the hepatocyte canalicular protein expression of AQP8, but not that of sinusoidal AQP9, is downregulated by a cholestatic dose of LPS. The decreased AQP8 protein expression is TNF-{alpha} mediated and seems to be caused by increased lysosomal and proteasomal protein degradation. The AQP8 downregulation was associated with decreased canalicular membrane water permeability, a mechanism that is likely to contribute to the molecular pathogenesis of LPS-induced cholestasis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grants PICT 05-10590 and 05-31670 (R. A. Marinelli) from Agencia Nacional de Promoción Científica y Tecnológica and by Grant PIP 6440 from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).


   ACKNOWLEDGMENTS
 
We thank Dr. N. F. LaRusso for encouragement and support, Dr. A. Vila for allowing us to make use of the stopped-flow spectrophotometer, and Dr. M. Tioni for assistance in these experiments. We also thank Dr. M. C. Larocca for critical reading of the manuscript and E. Ochoa for assistance in cell isolation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Marinelli, Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 570, 2000 Rosario, Santa Fe, Argentina (e-mail: rmarinel{at}unr.edu.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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