Abstract 238: Nuclear Renin-Angiotensin System in Renal Proximal Tubule Cells

Abstract

Compelling evidence for an intracellular renin-angiotensin system (RAS) is evident in multiple tissues including the heart, vasculature, brain and kidney. Indeed, the full complement of angiotensin receptors is localized on renal cortical nuclei; however, elucidation of the intracellular pathways that contribute to the expression of Ang II or Ang-(1-7) is incomplete. The current study sought to identify a proximal tubule epithelial cell model to facilitate our understanding of the regulation and function of this intracellular system. Utilizing the rat-derived NRK52E epithelial cells, immunofluorescent staining and western blots revealed nuclear expression of angiotensinogen (Aogen), renin and the prorenin receptor (PRR). Further characterization of Aogen expression utilizing an antibody against the protein region distal to Ang I ([des-Ang I]-Aogen) revealed a single 60 kDa band evident in the nuclear and the cytosolic fractions of the cells. A second antibody to the Ang I sequence of Aogen revealed expression in the cytosol and collected cell media, but not the nuclear fraction. The large molecular form of renin in the nuclear fraction suggests the predominance of prorenin in the nucleus. Basal renin activity in the nuclear fraction as measured by Ang I generation was significantly inhibited by the specific renin inhibitor aliskiren [770 ± 46 vs. 129 ± 14 pg Ang I/mg protein, n=3; p<0.01]. Conversion of the proform to active renin (total renin) by trypsin treatment of the nuclear fraction increased renin activity 2-fold [1933 ± 230 pg Ang I/mg; n=3; p<0.01]; aliskiren significantly reduced total renin activity by 92% [167 ± 24 pg Ang I/mg, n=3; p<0.01]. Utilizing specific radioimmunoassays, we detected both Ang II and Ang-(1-7) at concentrations of 50 ± 24 and 51 ± 39 pg/mg protein, respectively (n=3) in nuclear extracts of NRK cells. Collectively, these data suggest that processing of Aogen from either prorenin or renin may occur on the nucleus forming [des-Ang I]-Aogen and the active peptides Ang II and Ang-(1-7). We conclude that a nuclear prorenin/renin-Aogen pathway may constitute one source of peptide ligands for intracellular angiotensin receptors.