Abstract 020: A Mitochondrial Renin-Angiotensin System: Internalization of Angiotensinogen

There is compelling evidence for actions of an intracellular renin-angiotensin system (RAS) in various cell organelles including the endoplasmic reticulum, nucleus and the mitochondria (Mito). Indeed, angiotensin (Ang) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively in a previous study. Since elucidation of mitochondrial pathways for expression of RAS protein components as well as Ang II or Ang-(1-7) is equivocal at this time, we undertook a biochemical analysis of the Mito RAS from adult male sheep kidney. Cortical Mito were isolated by differential centrifugation and a discontinuous Percoll gradient. Purified Mito were co-enriched in the voltage-dependent anion channel, an outer Mito membrane marker as well as ATP synthase, an inner membrane marker. Angiotensinogen (Aogen; 55 kDa) was detected in Mito extracts by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the Ang I N-terminus. Two different renin antibodies identified a major 35 kDa protein band in the isolated Mito. Using the Ang I-directed Aogen antibody, active renin was confirmed by hydrolysis of Aogen that was abolished by aliskiren; however, trypsin exposure did not increase renin activity in the Mito. A pro-renin receptor (PRR) antibody failed to identify proteins in three Mito preparations, but revealed a prominent band in renal cortical membranes that corresponds to the size of PRR. Angiotensin peptides were quantified by three direct RIAs; the Mito content of Ang II and Ang-(1-7) were higher as compared to Ang I [23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; p<0.01, n=3]. Additionally, both neprilysin and thimet oligopeptidase activities that processed Ang I to Ang-(1-7) were evident. Finally, cortical Mito internalized radiolabeled Aogen at a rate of 33 ± 9 fmol/min/mg protein (n=3) at 37°C. The subsequent analysis of the labeled Mito by SDS-gel fractionation revealed a predominant radioactive band of 55 kDa for Aogen. Collectively, our data suggest that the internalization of Aogen and subsequent processing by active renin may yield des-[Ang I]-Aogen and the active peptides Ang II and Ang-(1-7) that may potentially contribute to mitochondrial function within the kidney.