ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway

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...

Source: AJP: Renal Physiology - December 11, 2016 Category: Urology & Nephrology Authors: Mamuya, F. A., Cano-Penalver, J. L., Li, W., Rodriguez Puyol, D., Rodriguez Puyol, M., Brown, D., de Frutos, S., Lu, H. A. J. Tags: CALL FOR PAPERS Source Type: research