Neprilysin

In the kidney, the endopeptidase neprilysin constitutes significant peptidase activity, particularly within the brush border region of the proximal tubules. Similar to ACE and ACE2, neprilysin is a zinc-dependent metallopeptidase that is anchored to the apical or extracellular region of the membrane, but is apparently resistant to enzymatic shedding. Although neprilysin was initially recognized for its enkephalin-degrading activity and frequently referred to as "enkephalinase", studies now reveal that this enzyme contributes to the metabolism of various peptides with cardiovascular actions including adrenomedullin, angiotensins, kinins, endothelins, substance P and the natriuretic peptides (Skidgel & Erdos 2004). Indeed, the development of neprilysin inhibitors, and more recently, dual or mixed inhibitors that target ACE as well remain potential therapies in cardiovascular disease (Veelken & Schmieder 2002). In general, these dual inhibitors were either equally or more effective in lowering blood pressure and reducing renal injury as compared to monotherapy with an ACE or neprilysin inhibitor (Kubota et al 2003; Tikkanen et al 2002). However, two large clinical trials (OCTAVE, OVERTURE) with the mixed inhibitor omapa-trilat revealed an increased incidence of angioedema. Moreover, the drug was no more effective than an ACE inhibitor alone (Kostis et al 2004; Packer et al 2002). A subsequent experimental study has shown that omapatrilat inhibits amniopeptidase P and, although less potent than its actions against ACE and neprilysin, this may further augment the local concentrations of kinins or substance P to exacerbate vascular permeability (Sulpizio et al 2005). In this aspect, the development of more selective inhibitors against ACE and neprilysin may be of clinical benefit.

The rationale for neprilysin inhibition primarily resides in preserving the "cardioprotective" peptides bradykinin and ANP or BNP. However, neprilysin readily metabolizes Ang II to the inactive fragment Ang-(1-4) which undergoes further hydrolysis to the dipeptides Asp-Arg and Val-Tyr. Neprilysin also cleaves endothelin, although it is not clear to what extent reduced intrarenal levels of endothelin are beneficial given the functional diversity of the endothelin A and B receptor subtypes within the kidney (Schiffrin 1999). The additional ACE inhibition would prevent the accumulation of Ang II and further contribute to the protection of kinins, as well as possibly reduce endothelin release. One possible caveat to this approach is that neprilysin is the major Ang-(1-7)-forming activity from Ang I or Ang-(1-9) in the circulation (Campbell et al 1998; Yamamoto et al 1992). Indeed, acute administration of the potent neprilysin inhibitor SCH3977 reduced circulating levels of Ang-(1-7) and increased blood pressure in the SHR chronically treated with the ACE inhibitor lisinoropril (Iyer et al 1997). Although plasma levels of neprilysin are low to non-detectable, the enzyme is appropriately localized to the ectocellular surface of endothelial and smooth muscle cells to contribute to the formation of Ang- (1-7) within the vasculature (Llorens-Cortes et al 1992).

In the kidney, neprilysin may contribute to both the formation as well as the degradation of the Ang-(1-7) (Allred et al 2000). Neprilysin cleaves the Pro7-Phe8

bond of Ang I to Ang-(1-7), but the very high levels of the enzyme in the kidney may continue to metabolize Ang-(1-7) at the Tyr5-Ile6 bond to form Ang-(1-4) and Ang-(5-7) (Allred et al 2000; Chappell et al 2001). Indeed, the mixed inhibitor omapatrilat augmented the urinary levels of Ang-(1-7) in both human hypertensives and the SHR model (Ferrario et al 2002a; Ferrario et al 2002b). The clinical study revealed a strong correlation between the reduction in blood pressure and increased excretion of Ang-(1-7) with the dual peptidase inhibitor (Ferrario et al 2002a). Interestingly, chronic treatment of male SHR with omapatrilat (2 weeks, 30 mg/kg daily) was also associated with the increased renal expression of ACE2. As shown in Fig. 5, immunocytochemical studies demonstrate enhanced expression

Angiotensine Neprilysin

Figure 5. Increased expression of Ang-(1-7) and ACE2 in the renal cortex of SHR following treatment with omapatrilat. Immunocytochemical staining for Ang-(1-7) in control (A) and treated (B) SHR; ACE2 staining in control (C) and treated (D), group. ACE2 staining in renal artery of treated SHR; arrow indicates intimal layer (E). Renal cortical ACE2 mRNA levels are significantly increased 2-fold following omapatrilat treatment (F); inset: ACE2 and EF-1a bands in the presence of the specific RT primers (RT+). Data are n = 7-8, mean ± SEM

Figure 5. Increased expression of Ang-(1-7) and ACE2 in the renal cortex of SHR following treatment with omapatrilat. Immunocytochemical staining for Ang-(1-7) in control (A) and treated (B) SHR; ACE2 staining in control (C) and treated (D), group. ACE2 staining in renal artery of treated SHR; arrow indicates intimal layer (E). Renal cortical ACE2 mRNA levels are significantly increased 2-fold following omapatrilat treatment (F); inset: ACE2 and EF-1a bands in the presence of the specific RT primers (RT+). Data are n = 7-8, mean ± SEM

of both ACE2 and Ang-(1-7) within the renal cortex of the treated-SHR (Chappell et al 2002). Omapatrilat treatment also revealed the renal vascular expression of ACE2 with staining evident in the intimal, medial and adventitial regions of the renal artery (Fig. 5E); vascular staining for the enzyme was undetectable in the untreated SHR group (Fig. 5C). Cortical mRNA of ACE2 expressed as a ratio to EF-1a increased 2-fold suggesting that transcriptional regulation contributes to the enhanced expression of ACE2 within the kidney (Fig. 5F). These studies are of interest as they reveal an additional mechanism of the vasopeptidase inhibitor that may result in the enhanced conversion of Ang II to Ang-(1-7) by ACE2, as well as protecting Ang-(1-7) from both neprilysin- and ACE-dependent degradation within in the kidney. Furthermore, these data suggest an important ability of the dual peptidase inhibitor (as well as the administration of other RAAS inhibitors alone) to restore ACE2 levels in the hypertensive kidney which may mitigate against the Ang II-ATj receptor axis of the RAAS. Indeed, Raizada and colleagues show that lenti-viral expression of ACE2 has amelioratory effects on blood pressure and cardiac fibrosis in the SHR, although the renal effects of enhanced enzyme activity were not ascertained (Diez-Freire et al 2006). Their data clearly demonstrate that ACE2 can markedly alter the balance of an activated RAAS pathway towards a normotensive phenotype. Further study is required to determine the extent that the beneficial actions of increased ACE2 reflect the greater inhibition of Ang II or the increased accumulation of Ang-(1-7) in the kidney or other tissue.

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