Superoxide via Sp3 Transcription Factor Up-Regulates Renal AT1 Receptor Function and Contributes to Hypertension
Hypertension is the most prevalent disease condition in the US affecting every 1 in 3 American adults. Renin angiotensin system (RAS) predominately regulates blood pressure and fluid homeostasis in the body. Angiotensin II type 1 (AT1) receptor is the key component of RAS that is implicated in hypertension. Oxidative stress is considered one of the main causes of hypertension. Studies including in our own lab showed that oxidative stress increases renal AT1 receptor function and contributes to hypertension. However, the exact mechanism of oxidative stress mediated increase in blood pressure is not completely known. Recent studies suggested that redox-sensitive transcription factors Sp3 and/or NF-κB regulate basal gene expression of AT1 receptor. Therefore, we designed studies to investigate the role of Sp3 and NF-κB in oxidative stress mediated hypertension or high blood pressure. We also designed studies to investigate the specificity of reactive oxygen species (superoxide vs. hydrogen peroxide) in regulating AT1 receptor function and high blood pressure. Results from cell culture studies (HK2 cells) demonstrated that SOD-inhibitor DETC but not hydrogen peroxide significantly increased fluorescence levels of superoxide probe dihydroethidium (DHE). Similarly, H2O2, but not DETC, treatment increased the fluorescence of the H2O2-sensitive probe dichloro-dihydro-fluorescein (DCFH). In addition, DETC, but not H2O2, increased the nuclear accumulation of Sp3 and NF-κB proteins. This effect was attenuated with tempol treatment. Furthermore, DETC increased AT1 receptor mRNA and protein expressions, which were attenuated with tempol treatment. However, H2O2 did not have any significant effect on AT1 mRNA expression. Results from transfection studies suggest that Sp3 plasmid increased while Sp3 siRNA decreased AT1 receptor protein expression. However, NF-κB plasmid failed to increase AT1 receptor protein expression. Immunofluorescence and biotinylation studies demonstrated that DETC treatment increased cell surface AT1 receptor. Moreover, angiotensin II increased PKC activity in vehicle-treated cells that further increased in DETC-treated cells, which was attenuated by AT1R blocker candesartan and SOD-mimetic tempol. Results from animal studies (SD rats) suggest that DETC treatment increased systolic and diastolic blood pressure, which were attenuated with tempol treatment. Furthermore, DETC treatment increased superoxide levels in kidney superficial cortex, which was attenuated with SOD-mimetic tempol. Candesartan infusion in DETC treated rats produced increased diuresis and natriuresis compared to controls. Tempol in DETC treated rats attenuated candesartan-induced increased diuresis and natriuresis. Importantly, lysine acetylation of Sp3 protein, an index of Sp3 activation, was significantly higher in DETC treated rats, which was attenuated with tempol treatment. Our data in both in vivo rat experiments and in vitro cell culture studies suggest that superoxide via Sp3 transcription factor up-regulates renal AT1 receptor function suggesting its role in hypertension. Specific targeting of superoxide and/or Sp3 may provide a selective and better therapeutic target to combat oxidative stress and/or its mediated effects responsible for hypertension and associated cardiovascular diseases.
AT1 receptors, Sp3 and NF-KB transcription factors, Superoxides, Hydrogen peroxide, Oxidative stress, Hypertension, HK2 cells, RAS, Tempol, DHE and DCFHDA
Networked Digital Library of Theses and Dissertations