Figure 4. ACE2 inhibition blocks the conversion of Ang II to Ang-(1-7) in isolated proximal tubules from female sheep. Sequential addition of peptidase inhibitors on the formation of Ang II metabolites include: AP, amniopeptidase (amastatin, bestatin); CHY, chymase, carboxypeptidase A (chymostatin, benzyl succinate); CY, cysteine protease (PCMB); NEP, neprilysin (SCH3977), ACE, angiotensin converting enzyme (lisinopril); ACE2 (MLN-4760). Data are n = 4, mean ± SEM
implications on the role of ACE as well, particularly whether ACE is involved in the formation (Li et al 2004) or degradation of Ang-(1-7) (Chappell et al 1998; Chappell et al 2000; Yamada et al 1998). Although Campbell and colleague demonstrate significant quantities of endogenous Ang-(1-9) in the rat kidney (Campbell et al 1991), chronic ACE inhibition or combined ACE/AT\ blockade (Chappell, unpublished observations) did not attenuate renal Ang-(1-7) levels in the rat. In addition, Ang-(1-7) levels within the kidney were maintained in tissue ACE knockout mice (Modrall et al 2003). Thus, these in vivo studies do not strongly support an ACE2-ACE cascade leading to the formation of Ang-(1-9) and Ang-(1-7) in the kidney.
The molecular studies utilizing ACE2 knockout mice provide additional evidence for the enzyme's role to balance the expression of Ang II and Ang-(1-7). We originally showed that ACE2 null mice exhibit higher circulating and tissue levels of Ang II (Crackower et al 2002). Indeed, the increased ratio of renal Ang II to Ang-(1-7) may contribute to the renal pathologies observed in older ACE2 null mice (Oudit et al 2006). Furthermore, the incidence of glomeruloscle-rosis and proteinuria in the male mice was markedly attenuated by AT1 receptor blockade. Several hypertensive models including the spontaneously hypertensive rat (SHR), stroke-prone SHR and Sabra salt sensitive rat exhibit lower mRNA levels and protein expression for ACE2 in the kidney than normotensive controls (Crackower et al 2002; Zhong et al 2004), as well as human prehypertensives (Keidar et al 2006). Tikellis and colleagues find that renal ACE2 expression is actually higher in the SHR than WKY normotensive controls at day one following birth, similar at 42 days and then dramatically declines in adult SHR by 80 days (Tikellis et al 2006). ACE activity, however, was markedly lower in the SHR kidney at all time points measured and declined in both strains at 80 days. Apart from the interesting pattern of development for ACE2 in the kidney, these data emphasize the need to at least consider alterations in both ACE and ACE2 in characterizing the functional output of the RAAS. Moreover, parallel studies to document the changes in renal Ang II and Ang-(1-7) during this developmental period are critical to establish the relevance to altered ACE and ACE2. It is clear that not all hypertensive models exhibit reduced ACE2 in the kidney. Our studies in the male mRen2.Lewis rat, a model of tissue renin expression with increased renal Ang II, found no difference in renal cortical ACE2 activity as compared to the normotensive Lewis strain, although cardiac activity was indeed lower in the hypertensive rats (Ferrario et al 2005a; Ferrario et al 2005b; Pendergrass et al 2006). Chronic blockade with either an ACE inhibitor or AT1 antagonist increased ACE2 activity in the kidneys of both the mRen2.Lewis and Lewis rats, but enzyme activity was significantly higher in the normotensive strain following treatment (Jessup et al 2006). This may reflect the situation where RAAS blockade does not completely reverse the extent of renal injury in the male mRen2.Lewis model. In this regard, the reduced ACE2 and elevated renal Ang II in the injured kidney of albumin-loaded rats was associated with increased NF-kB expression (Takase et al 2005). In contrast, ACE2 and its product Ang-(1-7) increase in the kidney of the rat during pregnancy (Brosnihan et al 2003). It is well known that the RAAS is activated during pregnancy, yet blood pressure is not altered in normal pregnancy, and it will be of interest to determine whether ACE2 expression within the kidney is altered with pre-eclampsia. Diabetic nephropathy is clearly dependent on an activated RAAS and both ACE inhibitors and AT1 receptor antagonists are effective in attenuating the progression of injury. Indeed, the renal expression of ACE2 is reduced in the proximal tubules of the streptozotocin-induced model of type I diabetes (Tikellis et al 2003; Wysocki et al 2006). Moreover, the attenuation of renal injury in this model by ACE inhibition is associated with increased ACE2 expression. A protective role for renal ACE2 is also evident from the findings that chronic ACE2 inhibition in the diabetic db/db mice exacerbates the extent of albuminuria almost 3-fold (Ye et al 2006). Although angiotensin content was not measured, the db/db mice exhibited increased glomerular expression of ACE and reduced ACE2 as compared to the control db/dm mice. Interestingly, the localization studies revealed distinct patterns of staining for ACE2 and ACE within the glomerulus - ACE2 in podocytes and ACE in the endothelial cells (Liebau et al 2006). Ang-(1-7) or the nonpeptide agonist AVE0991 attenuates proteinuria and improves renal vascular activity in the streptozoticin Type 1 diabetic rat, but did not reverse the urinary excretion of lysozyme, a marker of tubulointerstitial damage (Benter et al 2007). Moreover, the ratio of Ang-(1-7) to Ang II formed from exogenous Ang I was lower in glomeruli isolated from the kidneys of diabetic rats, however, the identity of the Ang-(1-7)-forming activity was not determined in this study (Singh et al 2005). Thus, in addition to the proximal tubule epithelium, the glomerulus may be a second key site within the kidney where ACE2 may influence the local expression of angiotensin peptides and renal function.
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