Expression and Localization of Aquaporin Water Channels in Rat Hepatocytes

Abstract

Although bile formation requires that large volumes of water be rapidly transported across liver epithelia, including hepatocytes, the molecular mechanisms by which water is secreted into bile are obscure. The aquaporins are a family of 10 channel-forming, integral membrane proteins of ϳ28 kDa numbered 0 -9 that allow water to rapidly traverse epithelial barriers in several organs including kidney, eye, and brain. We found transcripts of three of 10 aquaporins in hepatocytes (aquaporin 8 > > aquaporin 9 > aquaporin 0) by reverse transcription-polymerase chain reaction and quantitative ribonuclease protection assays; immunohistochemistry confirmed the presence of these three proteins in liver. Immunoblots of subcellular fractions of hepatocytes showed enrichment of aquaporins 0 and 8 in microsomes and canalicular plasma membranes; aquaporin 9 was enriched only in basolateral plasma membranes. Immunofluorescence of hepatocyte couplets confirmed the intracellular/canalicular localization of aquaporins 0 and 8 and the basolateral localization of aquaporin 9. Upon exposure of couplets to a choleretic stimulus (i.e. dibutyryl cAMP), aquaporin 8 redistributed to the canalicular plasma membrane; the subcellular distributions of aquaporins 0 and 9 were unaffected. In addition, exposure of couplets to dibutyryl cAMP caused an increase in canalicular water transport in the presence and absence of an osmotic gradient, an effect that was blocked by aquaporin inhibitors. These results provide evidence that aquaporins are present in hepatocytes and that aquaporins are involved in agonist-stimulated canalicular bile secretion.Primary bile is secreted by hepatocytes at the bile canaliculus and is modified via absorption and secretion of water, ions, and solutes by cholangiocytes, the epithelial cells that line the intrahepatic bile ducts. Hepatocytes are polarized epithelial cells that possess well defined canalicular and basolateral plasma membranes and are capable of rapidly transporting large volumes of water (1). Bile consists of 99% water, and water transport by hepatocytes is thought to occur passively in response to local, transient, osmotic gradients generated by the active transport of osmotically active solutes, especially bile acids (2). Two pathways exist by which water could potentially move from blood to bile across the hepatocyte epithelial barrier: a paracellular pathway through the tight junctions between adjacent hepatocytes and a transcellular pathway across hepatocytes. Furthermore, transcellular water movement across individual hepatocytes could theoretically occur either by diffusion through the lipid portion of the sinusoidal and canalicular hepatocyte plasma membranes or through aquaporin water channels, proteins that span the plasma membrane and allow for bi-directional, passive flux of water in response to soluteinduced osmotic gradients. The quantitative contributions of these potential pathways (i.e. paracellular versus transcellular) and mechanisms (i.e. diffusion versus channel-mediated) of water tr...

Aquaporin-1 Promotes Angiogenesis, Fibrosis, and Portal Hypertension Through Mechanisms Dependent on Osmotically Sensitive MicroRNAs

“…5 Briefly, mouse liver, mouse sinusoidal endothelial cells, or TSECs were homogenized in lysis buffer and cleared by centrifugation. Each sample, 50 to 100 g, was denatured, electrophoresed, transferred, blocked with milk, and incubated with antibodies to AQP1 (1:1000; ␣ Diagnostic International, San Antonio, TX), ␤-actin (1:10,000; Sigma, St. Louis, MO), or total extracellular signal-regulated kinase (1:1000; BD Biosciences, Franklin Lakes, NJ) overnight at 4°C.…”

“…5 Liver was harvested, sliced, embedded, and flash frozen on dry ice. Sections were cut to 4 to 8 m. Sections were fixed, quenched, blocked, and incubated with antibodies against AQP1 (1:500; ␣ Diagnostics International), CD31 (1:250), vWF (1:250), vascular endothelial growth factor receptor 2 (VEGFR2; 1:250), and endothelial nitric oxide synthase (eNOS; 1:250).…”

“…These proteins have well-characterized roles in epithelial secretion, absorption, and cell volume regulation. [5][6][7][8] More recently, they have also been implicated in localized protrusions of plasma membranes, cell motility, and angiogenesis. 9,10 Researchers 11-13 have demonstrated robust overexpression of AQP1 in both human and rodent chronic liver disease.…”

Lipopolysaccharide impairs hepatocyte ureagenesis from ammonia: Involvement of mitochondrial aquaporin‐8

“…In hepatocytes, AQP8 is also expressed at canalicular membranes [32,33] where it facilitates water transport during bile secretion [33][34][35]. In rats subjected to cecal ligation and puncture, or to LPS treatment, canalicular AQP8 is down-regulated by posttranslational mechanisms, i.e., increased protein degradation [18,36].…”

Aquaporin gene therapy for cholestasis

“…(4,5) In line with this is the assessment of the osmotic water permeability of hepatocyte basolateral and canalicular plasma membranes. (6) Studies in bile-canaliculi-forming, humanhepatocyte-derived HepG2 cells provide key additional support by demonstrating that the gene silencing of canalicular AQP8 markedly reduced osmotically driven and bile-agonist-induced canalicular water secretion.…”