Abstract
Superoxide (O2 (-)) exerts its physiological actions in part by causing changes in gene transcription. In thick ascending limbs flow-induced O2 (-)production is mediated byNADPHoxidase 4 (Nox4) and is dependent on protein kinase C (PKC). Polymerase delta interacting protein 2 (Poldip2) increases Nox4 activity, but it is not known whether Nox4 translocates to the nucleus and whether Poldip2 participates in this process. We hypothesized that luminal flow causes Nox4 translocation to the nuclei of thick ascending limbs in aPKC-dependent process facilitated by Poldip2. To test our hypothesis, we studied the subcellular localization of Nox4 and Poldip2 using confocal microscopy and O2 (-)production in the absence and presence of luminal flow. Luminal flow increased the ratio of nuclear to cytoplasmic intensity of Nox4 (N/C) from 0.3 ± 0.1 to 0.7 ± 0.1 (P < 0.01) and O2 (-)production from 89 ± 15 to 231 ± 16AU/s (P < 0.001). In the presence of flowPKCinhibition reduced N/C from 0.5 ± 0.1 to 0.2 ± 0.1 (P < 0.01). Flow-induced O2 (-)production was also blocked (flow: 142 ± 20AU/s; flow plusPKCinhibition 26 ± 12AU/s;P < 0.01). The cytoskeleton disruptor cytochalasin D (1 μmol/L) decreased flow-induced Nox4 translocation by 0.3 ± 0.01 (P < 0.01); however, it did not reduce flow-induced O2 (-) Flow did not alter Poldip2 localization. We conclude that: (1) luminal flow elicits Nox4 translocation to the nucleus in aPKC- and cytoskeleton-dependent process; (2) Nox4 activation occurs before translocation; and (3) Poldip2 is not involved in Nox4 nuclear translocation. Flow-induced Nox4 translocation to the nucleus may play a role in O2 (-)-dependent changes in thick ascending limbs.