Studies on angiotensin-(1-7) and mitochondria: The role of an intracellular renin-angiotensin system within the kidney

Several therapies we have available to treat high blood pressure and the resulting kidney failure target a classic circulating hormonal system - the renin-angiotensin System (RAS), and specifically, the ACE-Ang II-AT1 receptor axis. We have focused strategically on investigating novel intracellular pathways by which Ang-(1-7) is generated and degraded since this alternative pathway within the kidney and other tissues may antagonize the Ang II-AT1 receptor mediated actions. We recently identified the presence of a novel enzyme activity in the brain that degrades Ang-(1-7) and is negatively correlated with central Ang-(1-7) levels and blood pressure. Moreover, seminal studies as a part of my graduate work identified this enzyme activity in the kidney, specifically in the proximal tubules and in a human proximal tubule cell line. Utilizing the human tubule cells, we purified and identified the Ang-(1-7) degrading activity as Dipeptidyl-Peptidase 3 (DPP 3). Given the bioactive role of Ang-(1-7) counteracting the deleterious effects of Ang II, we further established that the beneficial intracellular actions of Ang-(1-7) may reflect the mitochondria. Thus, we demonstrated that mitochondria isolated from sheep renal cortex expressed RAS components and contained the molecular machinery to produce Ang-(1-7) intracellularly within the kidney that include the endopeptidases neprilysin (NEP) and thimet oligopeptidase (TOP) that process Ang I directly to Ang-(1-7).;We identified the presence of the RAS pathway precursor angiotensinogen (Aogen) in isolated kidney mitochondria, however it is unclear as to the extent that tubular Aogen reflects local synthesis or internalization within the kidney. Therefore, we sought to establish the extent that Aogen is internalized by proximal tubule cells and the intracellular pattern of distribution of the precursor. We developed a sensitive method to assess the cellular uptake of Aogen using a radiolabeled form of Aogen [125I-Aogen] and we determined that 125I-Aogen was internalized by proximal tubules at a rate of 0.002 +/- 0.0006 fmol/min/mg protein. We further established that approximately 20% of the internalized Aogen associated with the mitochondrial fraction of the proximal tubules. Moreover, we demonstrated that the isolated mitochondria from both sheep kidney and human proximal tubules internalize Aogen at a comparable rate suggesting that the uptake process of Aogen likely occurs in multiple species. Further characterization of Aogen internalization by human mitohchondria revealed that inhibiton of the mitochondrial membrane potential did not attenuate the uptake of the Aogen suggesting a novel and possibly energy-independent mechanism for mitochondrial transport.;Together these studies implicate a novel pathway for the delivery of the precursor protein Aogen into the renal tubules and the subsequent uptake by the mitochondria. Moreover, the demonstration of active renin and the NEP/TOP-Ang-(1-7)-Mas receptor axis within the renal mitochondria may portend for an important intracellular pathway to regulate sodium handling and blood pressure by Ang-(1-7). The mitochondrial Ang-(1-7) may also be regarded as a potential therapeutic target to maintain or augment Ang-(1-7) levels and preserve renal function.