*Systemic and Renal APA*

APA is a membrane-bound enzyme with a major presence in the kidney, in multiple tissues [31], and in a soluble form in the blood, probably due to the cleavage of membrane-bound APA [32]. APA, which thas also been called angiotensinase [33], transforms AngII, the most active systemic peptide, to AngIII, limiting the rate of angiotensin generation [34,35].

The importance of APA in BP control is evidenced by the BP reduction that follows the administration of purified APA [1] and by the BP increase induced by APA inhibitors [36]. In addition, APA-deficient mice are characterized by a high BP and an increased responsiveness to AngII [37]. These data support a role for APA in the regulation of BP under physiological conditions and in hypertensive humans and experimental models.

A study of SHR rats found that a decrease in kidney APA was related to an increase in RAS activity, whereas the administration of APA produced a dose-related reduction in systolic BP [38], showing a 2300-fold increase in activity in comparison to the AT1 blocker candesartan [39]. APA abnormalities have also been observed in the Goldblatt hypertension model [40]. Thus, Prieto et al. [41] reported decreased APA levels in the renal cortex of clipped and non-clipped kidneys in this model, suggesting the involvement of APA in augmenting the AngII-induced reabsorption of sodium and water. Renal RAS is also increased in Dahl salt-sensitive (DSS) rats [42]; in this model, age-related glomerular injury is associated with an increasing elevation of AngII levels because sclerotic glomeruli are less active in synthesizing APA [43]. In human subjects, serum APA activity increases in an age-dependent manner in both men and women [44], and this may be in relation to the metabolic clearance of AngII [45].

At the renal level, Velez et al. [46] observed an increased sensitivity to glomerular damage in APA-deficient BALB/c mice. The authors injected the APA-knockout (KO) mice with a nephrotoxic serum and observed glomerular hyalinosis and albuminuria at 96 h post-administration, whereas no renal injury was observed in the wild-type controls. Likewise, the 4-week infusion of AngII reduced podocyte nephrin levels in APA-KO mice but not in wild-type controls. These data indicate that the degradation of AngII induced by APA plays a protective role in glomerular injury.

Taken together, the above data indicate that an increase in systemic APA protects against hypertension. Conversely, a reduction in the activity of this enzyme maintains high levels of AngII and therefore promotes hypertension (Figure 2). As a conclusion, increased APA activity in the brain raises BP through an increased generation of AngIII, which is the main AT1 receptor agonist in the brain, whereas increased APA activity in the peripheral circulation lowers BP through the degradation of AngII (Figure 2).

#### **4. APN**

Aminopeptidase N (APN), also called leucine aminopeptidase and alanyl aminopeptidase, is a homodimeric, membrane-bound, zinc-dependent aminopeptidase [47]. APN cleaves AngIII to AngIV via the scission of arginine at the extreme N-terminus, which indicates its participation in the regulation of tissue and systemic RAS [48]. APN is highly expressed in the central nervous system and kidneys [47,49,50] and may develop multiple actions [47] besides peptide cleavage [51,52]. For this reason, APN is known as a "moonlighting protein" [53].

#### *4.1. Brain APN in BP Regulation*

Studies in rodents suggest the participation of brain APN in BP regulation [54]. Thus, icv administration of APN decreased BP in WKY and SHR animals, with more effect in the hypersensitive rats [55]. Conversely, icv administration of bestatin and amistatin, APN inhibitors, raised BP and induced a dipsogenic response in WKY and SHR animals (Figure 3) [55]. The paraventricular nucleus of the hypothalamus appears to be the target for APN in the brain, because its microinfusion at this site reduced BP in both SHR and WKY rats [56,57]. Central APN exerts its effects by transforming the pressor AngIII in the brain into AngIV. In this way, the pressor response induced by icv AngIII is potentiated by the APN antagonists bestatin and amistatin [38,58]. In line with these observations, administration of an angiotensin antagonist was found to inhibit the increase in BP produced by the icv administration of APN inhibitors [30,57].

**Figure 3.** Systemic and renal APA and APN in the control of blood pressure and renal function.
