Superoxide increases angiotensin II AT1 receptor function in human kidney‐2 cells

摘要

The redox‐sensitive Sp family transcription factor has been linked to the regulation of angiotensin II type 1 receptor (AT1R). However, the exact mechanism of AT1R regulation in renal cells is poorly understood. We tested the specificity of reactive oxygen species (ROS), superoxide vs. hydrogen peroxide (H2O2), and the specific role of Sp3 transcription factor, if any, in the regulation of AT1R in human kidney cells (HK2 cells). Superoxide dismutase (SOD) inhibitor diethyldithiocarbamate (DETC), but not H2O2 treatment, increased fluorescence levels of superoxide probe dihydroethidium (DHE). H2O2, but not DETC, treatment increased the fluorescence of the H2O2‐sensitive probe dichloro‐dihydro‐fluorescein (DCFH). These data suggest that SOD inhibition by DETC increases the superoxide but not H2O2 and exogenously added H2O2 is not converted to superoxide in renal cells. Furthermore, DETC, but not H2O2, treatment increased nuclear accumulation of Sp3, which was attenuated with the superoxide dismutase (SOD)‐mimetic tempol. DETC treatment also increased style="fixed-case">AT1R style="fixed-case">mRNA and protein levels that were attenuated with tempol, whereas H2O2 did not have any effects on style="fixed-case">AT1R style="fixed-case">mRNA. Moreover, Sp3 overexpression increased, while Sp3 depletion by si style="fixed-case">RNA decreased, protein levels of style="fixed-case">AT1R. In addition, Sp3 si style="fixed-case">RNA in the presence of style="fixed-case">DETC decreased style="fixed-case">AT1R protein expression. Furthermore, style="fixed-case">DETC treatment increased the levels of cell surface style="fixed-case">AT1R as measured by biotinylation and immunofluorescence studies. Angiotensin style="fixed-case">II increased style="fixed-case">PKC activity in vehicle‐treated cells that further increased in style="fixed-case">DETC‐treated cells, which was attenuated by style="fixed-case">AT1R blocker candesartan and style="fixed-case">SOD‐mimetic tempol. Taken together, our results suggest that superoxide, but not H₂O₂, via Sp3 up‐regulates style="fixed-case">AT1R expression and function in the renal cells.