Abstract 124: Luminal Flow Induces NADPH Oxidase 4 Translocation to the Nuclei of Thick Ascending Limbs
Superoxide (O2-) exerts its physiological and pathophysiological actions in part by causing changes in gene transcription. In the thick ascending limb of the loop of Henle (TAL), luminal flow-induced O2- production is mediated by NADPH oxidase 4 (NOX4). Recently, polymerase (DNA-directed), delta interacting protein 2 (poldip2) has been shown to increase NOX4 activity. However, it is not known whether NOX4 translocates to the nucleus when activated or whether poldip2 participates in this process. We hypothesized that luminal flow causes translocation of NOX4 to the nucleus of the TAL facilitating its interaction with poldip2 in a protein kinase C (PKC)-dependent process. To test our hypothesis, we studied the subcellular localization of NOX4 and poldip2 using confocal microscopy and measured O2– production. Isolated TALs were studied in the absence and presence of luminal flow. To disrupt the cytoskeleton and inhibit PKC we used cytochalasin D (1uM) and staurosporine (10nM), respectively. Luminal flow increased the amount of NOX4 in the nucleus from 4±1 arbitrary units (AU) to 31±1 AU (p <0.0001; n=6). This redistribution was blocked by cytochalasin D (7±2, p < 0.04 vs. controls; n=6). Similarly, staurosporine decreased the intensity of NOX4 within the nuclear region of perfused TALs (3±2 AU vs controls, p < 0.01; n=5), Luminal flow increased poldip2 in the nucleus from 18±1 AU (n=3) to 29±1 AU (p<0.01; n=6). Net O2- production increased in response to luminal flow from 89±15 AU/S to 231±16 AU/S (p < 0.001; n=6). Inhibiting PKC decreased flow-induced O2– production in isolated TALs. However, disrupting the actin cytoskeleton with cytochalasin D did not block flow-induced O2– production (no flow + cytochalasin: 135± 19 vs. flow + cytochalasin: 285 ± 41 AU; p <0.04; n=7). We conclude that NOX4 translocates to the nucleus when activated by luminal flow, as does poldip2. Translocation is dependent on the actin cytoskeleton but O2– production is not. In contrast PKC mediates both processes. Translocation of NOX4 to the nucleus may facilitate changes in gene transcription caused by O2–.
Author Disclosures: F. Saez: None. J.L. Garvin: None.
This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
- © 2015 by American Heart Association, Inc.