Abstract 572: Transcriptional Regulation Of Renal Dopamine D1 Receptor Function During Oxidative Stress
There exists a strong link between Oxidative stress, renal dopaminergic system and hypertension. It is reported that reactive oxygen species attenuate renal proximal tubular dopamine receptor (D1R) function which disrupts sodium regulation and leads to hypertension. The mechanisms for renal D1R dysfunction, however are not clear. Here in, we investigated the role of redox sensitive transcription factors AP1 and SP3 in transcriptional suppression of D1R gene function and subsequent D1R signaling. Human kidney proximal tubular (HK2) cell were treated with a pro-oxidant L-buthionine sulfoximine (BSO, 50 mM for 72 hr) with and without a polyphenol resveratrol (250 μM). Treatment of HK2 cells with BSO caused oxidative stress (malondialdehyde [MDA], nmol/mg protein: control: 0.13 ± 0.01, BSO: 0.20 ± .02) and significantly reduced D1R mRNA and membrane receptor number (fmol/mg protein; control—140.1 ± 5.6, BSO—81.7 ± 4.4). Incubation of HK2 cells with SKF38393, a D1R agonist, caused a concentration dependent inhibition of Na/K-ATPase (86Rb uptake, nmol/mg protein, control: basal—9.2 ± 0.2, SKF (1 μM)—6.3 ± 0.1). However, SKF38393 failed to inhibit Na/K-ATPase in BSO-treated cells (BSO: basal—9.5 ± 0.3, SKF—8.6 ± 0.2). Cells incubated with BSO exhibited increased AP1 (c-fos) and SP3 nuclear expression. Resveratrol per se had no effect on oxidative milieu (MDA: 0.11 ± 0.01) or D1R expression (143.3 ± 4.4), but it mitigated BSO-induced oxidative stress (MDA: 0.13 ± 0.02), AP1—SP3 up-regulation and D1R down-regulation (133.6 ± 6.2) and dysfunction (resveratrol + BSO [86Rb uptake]: basal—9.7 ±0.3, SKF—6.7 ± 0.4). Incubation of cells with AP1 or SP3 specific siRNA reduced the expression of respective protein by ~85% and abolished BSO-induced D1R dysfunction. Down-regulation of SP3 had no effect on BSO-mediated AP1 activation, however, AP1 siRNA blocked BSO-dependent SP3 up-regulation. In cells transfected with Nrf2 siRNA, resveratrol failed to block BSO-induced AP1—SP3 activation and D1R dysfunction. These data shows that oxidative stress
via AP1—SP3 activation suppresses D1R transcription which leads to D1R dysfunction. Resveratrol activates Nrf2, mitigates oxidative stress, blocks AP1—SP3 signaling and prevents D1R down-regulation and dysfunction.
Author Disclosures: A. Banday: None. M.F. Lokhandwala: None.
- © 2014 by American Heart Association, Inc.