Evidence for Protein Kinase C Dependent Stimulation of Reactive Oxygen Species in Isolated Nuclei of Renal Epithelial Cells

We previously reported evidence for functional angiotensin (Ang) receptors in isolated nuclei of the rat kidney. Ang II stimulates AT1 receptors on nuclei to increase the formation of reactive oxygen species (ROS) through a protein kinase C (PKC) pathway. To identify a potential cell model for this intracellular system, we examined ROS production and PKC isoform expression in the NRK52E cell line derived from rat renal proximal tubules. Nuclei were isolated from confluent cells by hypoosmotic shock, differential centrifugation and subsequent separation through 0.8M sucrose. To measure ROS, nuclei were loaded with the dichlorofluorescein (DCF, 10 μM) in the absence or presence of either the H2O2 scavenger ebselen (10 μM), an NADPH oxidase inhibitor DPI (10 μM) and the PKC inhibitor Bisindolylmaleimide I (PKCi, 10 μM). Nuclei were then exposed to the PKC agonist phorbol myristate acetate (PMA, 1 μM) for 10 min and the fluorescence measured in a plate reader. PMA significantly increased ROS production (79 + 21%, n=5) above the control nuclei. The PMA stimulation was essentially abolished by both ebselen (14 + 7%) and the PKCi (−2.0 ± 25%) but partially inhibited by DPI [46 ± 13%]. Immunoblots of nuclei revealed both PKC‐α; and PKC‐δ; isoforms. In summary, we demonstrate that isolated nuclei from NRK52E cells contain a functional PKC system that is coupled to the formation of ROS.