Guillermo L. Lehmann

Aquaporin Water Channels in Central Nervous System

Aquaporins (AQPs) are a family of water-selective channels that provide a major pathway for osmotically driven water transport through cell membranes. Some members of the aquaporin family have been identified in the central nervous system (CNS). The water channel aquaporin 1 (AQP1) is restricted to the apical domain of the choroid plexus epithelial cells. The AQP4 is abundantly expressed in astrocyte foot processes and ependymocytes facing capillaries and brain-cerebrospinal fluid (CSF) interfaces, whereas AQP9 is localized in tanycytes and astrocytes processes. The mRNA for other aquaporin homologs (i.e., AQP3, 5, and 8) have been recently found in cultured astrocytes. Based on their subcellular localization and data obtained from functional studies, it is assumed that aquaporins are implicated in water movements in nervous tissue and may play a role in central osmoreception, K+ siphoning, and cerebrospinal fluid formation. There have been recent reports describing different aquaporin-responses under pathologic states leading to brain edema. The data available provide a better understanding of the mechanisms responsible for brain edema and indicate that aquaporins are potential targets for drug development.

Activation of Trpv4 reduces the hyperproliferative phenotype of cystic cholangiocytes from an animal model of ARPKD

BACKGROUND & AIMS In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes. METHODS Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats. RESULTS Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts. CONCLUSIONS Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.

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

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 (P(f)) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (65 +/- 1 vs. 49 +/- 1 microm/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.

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 approximately 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.

Phosphoinositide 3-kinase is involved in the glucagon-induced translocation of aquaporin-8 to hepatocyte plasma membrane

Background information. PI3K (phosphoinositide 3‐kinase) mediates several signal transduction pathways in hepatocytes, including some involved in the regulation of vesicle trafficking. Hepatocytes express the water channel AQP8 (aquaporin‐8) predominantly in an intracellular location, and it redistributes to the canalicular membrane, upon stimulation with the hormone glucagon, by a cAMP/protein kinase A‐dependent mechanism. Since glucagon is capable of stimulating PI3K activity in hepatocytes and a cross talk between cAMP and PI3K has been suggested, in the present study, we examine whether PI3K activation is involved in the glucagon‐induced translocation of AQP8.

Peritoneal sepsis downregulates liver expression of Aquaporin‐8: a water channel involved in bile secretion

To the Editor: Cholestasis is a common feature of sepsis (1), and its pathogenesis cannot be understood without a thorough understanding of the molecular basis of bile formation. The active secretion of solutes into the biliary space is the principal driving force for bile formation, which results in the secondary influx of water in response to the osmotic gradient created. Aquaporins (AQPs), a family of channel proteins that facilitates the osmotic water transport across cell membranes (2), determine the water permeability of hepatocyte plasma membrane domains. Thus, AQP8 would modulate the canalicular, rate-limiting water flow, while AQP9 would contribute to the sinusoidal water uptake during hepatocyte bile formation (3–5). Sepsis-induced cholestasis is thought to be mediated by endotoxins, which are outer bacterial membrane lipopolysaccharides (LPS), and by LPSinduced cytokines such as tumour necrosis factor (TNF)a and interleukins from Kupffer cells. This syndrome is most commonly associated with Gram-negative sepsis, but it has also been reported in association with polymicrobial infections. Cytokines seem to act directly on hepatocytes causing dysfunction in bile formation (1). Although the precise molecular mechanisms of cholestasis have not yet been elucidated, it is thought that defective expression of key solute canalicular membrane transporters (e.g. the bile salt export pump Bsep and organic anion transporter Mrp2) is directly involved (6). Because bile consists of more than 95% water, it seems probably that the molecular and regulatory aspects involved in water transport can also be implicated. In an LPS-induced cholestasis

Hepatic Gene Transfer of Human Aquaporin-1 Improves Bile Salt Secretory Failure in Rats With Estrogen-Induced Cholestasis

The adenoviral gene transfer of human aquaporin‐1 (hAQP1) water channels to the liver of 17α‐ethinylestradiol‐induced cholestatic rats improves bile flow, in part by enhancing canalicular hAQP1‐mediated osmotic water secretion. To gain insight into the mechanisms of 17α‐ethinylestradiol cholestasis improvement, we studied the biliary output of bile salts (BS) and the functional expression of the canalicular BS export pump (BSEP; ABCB11). Adenovector encoding hAQP1 (AdhAQP1) or control vector was administered by retrograde intrabiliary infusion. AdhAQP1‐transduced cholestatic rats increased the biliary output of major endogenous BS (50%‐80%, P < 0.05) as well as that of taurocholate administered in choleretic or trace radiolabel amounts (around 60%, P < 0.05). Moreover, liver transduction with AdhAQP1 normalized serum BS levels, otherwise markedly elevated in cholestatic animals. AdhAQP1 treatment was unable to improve BSEP protein expression in cholestasis; however, its transport activity, assessed by adenosine triphosphate‐dependent taurocholate transport in canalicular membrane vesicles, was induced by 90% (P < 0.05). AdhAQP1 administration in noncholestatic rats induced no significant changes in either biliary BS output or BSEP activity. Canalicular BSEP, mostly present in raft (high cholesterol) microdomains in control rats, was largely found in nonraft (low cholesterol) microdomains in cholestasis. Considering that BSEP activity directly depends on canalicular membrane cholesterol content, decreased BSEP presence in rafts may contribute to BSEP activity decline in 17α‐ethinylestradiol cholestasis. In AdhAQP1‐transduced cholestatic rats, BSEP showed a canalicular microdomain distribution similar to that of control rats, which provides an explanation for the improved BSEP activity. Conclusion: Hepatocyte canalicular expression of hAQP1 through adenoviral gene transfer promotes biliary BS output by modulating BSEP activity in estrogen‐induced cholestasis, a novel finding that might help us to better understand and treat cholestatic disorders. (Hepatology 2016;64:535‐548)