Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D₂ receptor-induced inhibition of NADPH oxidase

The dopamine D₂ receptor (D₂R) regulates renal reactive oxygen species (ROS) production, and impaired D₂R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D₂R may decrease ROS production, in part, through regulation of PON2. D₂R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D₂^-/- relative to D ₂^/ mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6 mm Hg; diastolic, 97±4 vs 65±7 mm Hg; mean 113±4 vs 75±7 mm Hg). To determine the relevance of the PON2 and D₂R interaction in humans, we studied human renal proximal tubule cells. Both D₂R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D₂R/D₃R agonist quinpirole (1 μM, 24 h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24 h. Silencing PON2 (siRNA, 10 nM, 48 h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10 μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D₂R and may, in part, mediate the inhibitory effect of renal D₂R on NADPH oxidase activity and ROS production.