Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60–70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of ∼70% ( P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (−65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (−80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.

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Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units

AJP Cell Physiology ◽

10.1152/ajpcell.00162.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. C338-C346 ◽

Cited By ~ 31

Author(s):

Ai-Yu Gong ◽

Anatoly I. Masyuk ◽

Patrick L. Splinter ◽

Robert C. Huebert ◽

Pamela S. Tietz ◽

...

Keyword(s):

Bile Duct ◽

Water Transport ◽

Water Movement ◽

Water Channel ◽

Intrahepatic Bile Duct ◽

Enzymatic Digestion ◽

Bile Formation ◽

Initial Application ◽

Luminal Area ◽

Water Secretion

We previously reported the development of reproducible techniques for isolating and perfusing intact intrahepatic bile duct units (IBDUs) from rats. Given the advantages of transgenic and knockout mice for exploring ductal bile formation, we report here the adaptation of those techniques to mice and their initial application to the study of water transport across mouse intrahepatic biliary epithelia. IBDUs were isolated from livers of normal mice by microdissection combined with enzymatic digestion. After culture, isolated IBDUs sealed to form intact, polarized compartments, and a microperfusion system employing those isolated IBDUs developed. A quantitative image analysis technique was used to observe a rapid increase of luminal area when sealed IBDUs were exposed to a series of inward osmotic gradients reflecting net water secretion; the choleretic agonists secretin and forskolin also induced water secretion into IBDUs. The increase of IBDU luminal area induced by inward osmotic gradients and choleretic agonists was reversibly inhibited by HgCl2, a water channel inhibitor. With the use of a quantitative epifluorescence technique in perfused mouse IBDUs, a high osmotic water permeability ( P f = 2.5–5.6 × 10−2 cm/s) was found in response to osmotic gradients, further supporting the presence of water channels. These findings suggest that, as in the rat, water transport across intrahepatic biliary epithelia in mice is water channel mediated.

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Reduced osmotic water permeability of the peritoneal barrier in aquaporin-1 knockout mice

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.1.c76 ◽

1999 ◽

Vol 276 (1) ◽

pp. C76-C81 ◽

Cited By ~ 101

Author(s):

Baoxue Yang ◽

Hans G. Folkesson ◽

Jian Yang ◽

Michael A. Matthay ◽

Tonghui Ma ◽

...

Keyword(s):

Water Transport ◽

Peritoneal Cavity ◽

Knockout Mice ◽

Fluid Transport ◽

Water Movement ◽

Osmotic Gradient ◽

Half Time ◽

Aquaporin 1 ◽

Osmotic Water ◽

Major Route

Aquaporin-1 (AQP1) water channels are expressed widely in epithelia and capillary endothelia involved in fluid transport. To test whether AQP1 facilitates water movement from capillaries into the peritoneal cavity, osmotically induced water transport rates were compared in AQP1 knockout [(−/−)], heterozygous [(+/−)], and wild-type [(+/+)] mice. In (+/+) mice, RT-PCR showed detectable transcripts for AQP1, AQP3, AQP4, AQP7, and AQP8. Immunofluorescence showed AQP1 protein in capillary endothelia and mesangium near the peritoneal surface and AQP4 in adherent muscle plasmalemma. For measurement of water transport, 2 ml of saline containing 300 mM sucrose (600 mosM) were infused rapidly into the peritoneal cavity via a catheter. Serial fluid samples (50 μl) were withdrawn over 60 min, with albumin as a volume marker. The albumin dilution data showed significantly decreased initial volume influx in AQP1 (−/−) mice: 101 ± 8, 107 ± 5, and 42 ± 4 (SE) μl/min in (+/+), (+/−), and (−/−) mice, respectively [ n = 6–10, P < 0.001, (−/−) vs. others]. Volume influx for AQP4 knockout mice was 100 ± 8 μl/min. In the absence of an osmotic gradient,3H2O uptake [half time = 2.3 and 2.2 min in (+/+) and (−/−) mice, respectively], [14C]urea uptake [half time = 7.9 and 7.7 min in (+/+) and (−/−) mice, respectively], and spontaneous isosmolar fluid absorption from the peritoneal cavity [0.47 ± 0.05 and 0.46 ± 0.04 ml/h in (+/+) and (−/−) mice, respectively] were not affected by AQP1 deletion. Therefore, AQP1 provides a major route for osmotically driven water transport across the peritoneal barrier in peritoneal dialysis.

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Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogen-induced cholestasis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00386.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. G905-G912 ◽

Cited By ~ 31

Author(s):

Flavia I. Carreras ◽

Guillermo L. Lehmann ◽

Domenico Ferri ◽

Mariana F. Tioni ◽

Giuseppe Calamita ◽

...

Keyword(s):

Water Permeability ◽

Protein Level ◽

Intrahepatic Cholestasis ◽

Rat Hepatocytes ◽

Functional Expression ◽

Bile Secretion ◽

Northern Blotting ◽

Bile Formation ◽

Canalicular Membrane ◽

Lysosomal Protease

Our previous work supports a role for aquaporin-8 (AQP8) water channels in rat hepatocyte bile formation mainly by facilitating the osmotically driven canalicular secretion of water. In this study, we tested whether a condition with compromised canalicular bile secretion, i.e., the estrogen-induced intrahepatic cholestasis, displays defective hepatocyte AQP8 functional expression. After 17α-ethinylestradiol administration (5 mg·kg body wt−1·day−1 for 5 days) to rats, the bile flow was reduced by 58% ( P < 0.05). 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, 17α-ethinylestradiol-induced cholestasis did not significantly affect the protein level or the subcellular localization of sinusoidal AQP9. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability ( Pf) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 ± 1 vs. 57 ± 2 μm/s) in cholestasis, consistent with defective canalicular AQP8 functional expression. By Northern blotting, we found that AQP8 mRNA expression was increased by 115% in cholestasis, suggesting a posttranscriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 downregulation. In conclusion, hepatocyte AQP8 protein expression is downregulated in estrogen-induced intrahepatic cholestasis, presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated with impaired osmotic membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP8 contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.

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Water transport in neonatal and adult rabbit proximal tubules

AJP Renal Physiology ◽

10.1152/ajprenal.00341.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. F280-F285 ◽

Cited By ~ 8

Author(s):

Raymond Quigley ◽

Michel Baum

Keyword(s):

Water Transport ◽

Water Permeability ◽

Water Movement ◽

Chloride Transport ◽

Basolateral Membrane ◽

Membrane Vesicles ◽

Proximal Tubules ◽

Adult Rabbit ◽

Osmotic Gradient ◽

Intracellular Compartment

We have recently demonstrated that although the osmotic water permeability ( P f) of neonatal proximal tubules is higher than that of adult tubules, the P f of brush-border and basolateral membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal convoluted tubules (PCTs) in neonatal (9–16 days old) and adult rabbits to determine whether the intracellular compartment or paracellular pathway is responsible for the maturational difference in transepithelial water transport. The permeability of n-butanol was higher in the neonatal PCT than the adult PCT at all temperatures examined, whereas the diffusional water permeability was identical. Increasing the osmotic gradient increased volume absorption in both the neonatal and the adult PCT to the same degree. The P f was not different between the neonatal and the adult PCT at any osmotic gradient studied. To assess solvent drag as a measure of the paracellular transport of water, the effect of the osmotic gradient on mannitol and chloride transport were measured. There was no change in chloride or mannitol transport with the increased osmotic gradient in either group, indicating that there was no detectable paracellular water movement. In addition, the mannitol permeability of the neonatal PCT was found to be lower than that of the adult PCT with the isotonic bath (8.97 ± 4.01 vs. 40.49 ± 13.89 μm/s, P < 0.05). Thus the intracellular compartment of the neonatal PCT has a lower resistance for water transport than the adult PCT and is responsible for the higher than expected P f in the neonatal PCT.

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OSMOTIC INHIBITION OF THE NATRIFERIC RESPONSE OF THE TOAD URINARY BLADDER TO VASOPRESSIN

Journal of Endocrinology ◽

10.1677/joe.0.0670119 ◽

1975 ◽

Vol 67 (1) ◽

pp. 119-125

Author(s):

P. J. BENTLEY

Keyword(s):

Urinary Bladder ◽

Water Movement ◽

Short Circuit ◽

Electrical Potential ◽

Short Circuit Current ◽

Toad Bladder ◽

Toad Urinary Bladder ◽

Electrical Potential Difference ◽

Osmotic Gradient

SUMMARY The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.

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