title: ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway.
abstract: Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, to open a path toward effective diabetes insipidus therapeutics. A recent study has shown that integrin-linked kinase (ILK) conditional-knockdown mice developed polyuria along with decreased AQP2 expression. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway.
authors.affiliation: Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Fahmy A
authors.initials: FA
authors.lastname: Mamuya
authors.affiliation: Department of Systems Biology, Physiology Unit, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain; Instituto Reina Sofia de Investigación Renal and Red de Investigación Renal (REDinREN), Instituto de Salud Carlos III, Madrid, Spain; and.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Cano-Peñalver
authors.affiliation: Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Biomedical Research Foundation and Nephrology Department, Hospital Príncipe de Asturias, Alcalá de Henares, and Instituto Reina Sofia de Investigación Renal and REDinREN from Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: Rodriguez Puyol
authors.affiliation: Department of Systems Biology, Physiology Unit, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain; Instituto Reina Sofia de Investigación Renal and Red de Investigación Renal (REDinREN), Instituto de Salud Carlos III, Madrid, Spain; and.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rodriguez Puyol
authors.affiliation: Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
authors.affiliation: Department of Systems Biology, Physiology Unit, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain; Instituto Reina Sofia de Investigación Renal and Red de Investigación Renal (REDinREN), Instituto de Salud Carlos III, Madrid, Spain; and.
authors.forename: Sergio
authors.initials: S
authors.lastname: de Frutos
authors.affiliation: Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; Lu.Hua@mgh.harvard.edu.
authors.forename: Hua Ann Jenny
authors.initials: HA
authors.lastname: Lu
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000099200:Scaffold Protein ILK
chemical_list: D051399:Aquaporin 2; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK
keywords: aquaporin-2; diabetes insipidus; integrin-linked kinase; kidney tubular cells