**2. Renin**

angiotensin-converting enzyme (ACE) (**Figure 1**). Ang II binds to the angiotensin type 1 receptor (AT1R) to mediate both peripheral and central mechanisms in the regulation of blood pressure. Activation of the ACE-Ang II-AT1R pathway is also associated with various pathological responses including fibrosis, inflammation, metabolic dysregulation, heart failure, cancer, aging, and diabetic injury [1–5]. Indeed, the targeting of this pathway by biochemical approaches that block the formation of Ang II through ACE inhibitors or binding of Ang II to the AT1R by receptor antagonists is an effective therapy for the treatment of cardiovascular disease. It is now evident that these therapies have revealed alternative pathways within the RAS that may contribute to the beneficial actions of the RAS blockade. For example, the targeting of ACE reduces Ang II expression, but markedly enhances the circulating levels of Ang-(1-7), a ligand for the AT7/MasR that generally opposes the actions of the Ang II-AT1R pathway [6, 7]. The chronic treatment with AT1R antagonists may also increase the formation of Ang-(1-7) through ACE2, as well as shunt Ang II to the AT2R pathway that shares similar properties to the Ang-(1-7) system [8, 9]. Since the RAS is now viewed as a more complex array of components that can be functionally partitioned into distinct receptors and peptide ligands, we present an overview of the peptidases the may constitute the alternative Ang-(1-7) axis of the RAS. We include a discussion of renin, ACE, and chymase as they pertain to the formation of the substrates Ang I and Ang II for the direct processing to

**Figure 1.** Processing cascade for angiotensin-(1-7). Renin cleaves angiotensinogen to Ang I which is further processed to the biologically active peptides Ang II by ACE or chymase (CHYM). Ang II undergoes further processing by the carboxypeptidase ACE2 to form Ang-(1-7). Ang-(1-7) is formed directly through non-Ang II pathways by the direct processing of Ang I by the endopeptidases neprilysin (NEP) and thimet oligopeptidase (TOP). Ang-(1-7) is subsequently metabolized by ACE to Ang-(1-5) and dipeptidyl peptidase 3 (DPP3) to Ang-(3-7) and Ang-(5-7). Adapted

Ang-(1-7), as the well as the role of ACE to metabolize Ang-(1-7).

from Chappell [54].

4 Enzyme Inhibitors and Activators

Renin [EC 3.4.23.15] belongs to the family of aspartyl-type acid proteases (molecular size: 30– 40 kDa); however, the protease exhibits a more neutral pH optima of 6.5–8.0. The only known substrate for renin is angiotensinogen, and renin cleaves off the N-terminal portion of angiotensinogen to form the inactive peptide Ang I. The formation of Ang I is considered the enzymatic event that initiates the RAS cascade (**Figure 1**). The enzyme is synthesized predominantly in the juxtaglomerular (JG) cells of the kidney and is stored in both inactive (prorenin) and active forms for subsequent release into the afferent arteriole to reach the circulation. The collecting duct (CD) cells are another source of renin within the kidney that primarily secretes the active form of renin from the apical aspect of the principal cells into the tubular fluid [10, 11]. The distal secretion of renin into the tubular fluid would presumably contribute to the processing of angiotensinogen that may arise from either proximal tubule synthesis or apical release or the glomerular filtration of the protein. Interestingly, the regulation of JG renin appears to be distinct from CD renin as Ang II reduces JG renin release and synthesis constituting the classic short-loop negative feedback system for RAS activation, whereas the peptide stimulates CD renin release suggesting a positive feedback system for renin in the distal nephron [12, 13].

There is evidence for alternative gene products of renin expressed in the kidney, brain, heart, and adrenal gland [14–17]. These products arise from an alternative start site for renin transcription that is situated downstream from the typical start site and yields a shorter, but active form of the enzyme. The truncated form of renin lacks the secretory signal of the protein and the enzyme is not secreted, but resides within the cell. Peters and colleagues find that truncated renin specifically localized to the mitochondria and that the active form of renin, but not prorenin, was internalized by mitochondria [14, 18, 19]. The overexpression of the active renin isoform protected the cells under high glucose conditions and this effect was not reversed by the renin inhibitor aliskerin or an AT1R antagonist suggesting that the beneficial effects may not reflect activation of the Ang II-AT1R axis [20]. In support of these findings, we recently reported the presence of active renin in isolated mitochondria from the sheep renal cortex [21]. In this case, we also quantified the mitochondrial levels of both Ang II and Ang-(1-7) (50–60 fmol/mg protein), which may indicate that the intracellular form of renin contributes to the cellular expression of angiotensin peptides within the kidney [21]. In the renal NRK-52 epithelial cell line, immunocytochemical staining for renin was evident in the nucleus of these cells [22]. Renin activity as measured by aliskerin-sensitive inhibition of Ang I formation from angiotensinogen was also evident in isolated nuclei in the renal epithelial NRK-52 cells, as well as quantifiable levels of Ang I, Ang II, and Ang-(1-7) [22]. In confirmation of intracellular renin within the kidney, Ishigami and colleagues report a truncated renin transcript expressed in the proximal tubules of the mouse kidney [16]. Overexpression of the renin isoform within the proximal tubules was associated with a sustained elevation in blood pressure and no change in the circulating levels of renin [16]. Although the intracellular distribution of the renin isoform was not ascertained, the fact that overexpression of tubular renin augmented blood pressure is consistent with previous studies demonstrating that the increased expression of proximal tubule angiotensinogen, the AT1R, or intracellular Ang II also resulted in a sustained increase in blood pressure [23–28].
