Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

Abstract

The renin-angiotensin system (RAS) constitutes a key hormonal system in the physiological regulation of blood pressure through peripheral and central mechanisms. Indeed, dysregulation of the RAS is considered a major factor in the development of cardiovascular pathologies, and pharmacological blockade of this system by the inhibition of angiotensin-converting enzyme (ACE) or antagonism of the angiotensin type 1 receptor (AT1R) offers an effective therapeutic regimen. The RAS is now defined as a system composed of different angiotensin peptides with diverse biological actions mediated by distinct receptor subtypes. The classic RAS comprises the ACE-ANG II-AT1R axis that promotes vasoconstriction; water intake; sodium retention; and increased oxidative stress, fibrosis, cellular growth, and inflammation. In contrast, the nonclassical RAS composed primarily of the ANG II/ANG III-AT2R and the ACE2-ANG-(1-7)-AT7R pathways generally opposes the actions of a stimulated ANG II-AT1R axis. In lieu of the complex and multifunctional aspects of this system, as well as increased concerns on the reproducibility among laboratories, a critical assessment is provided on the current biochemical approaches to characterize and define the various components that ultimately reflect the status of the RAS.

Keywords: ACE; angiotensin; heart; renin.

Fig. 1.

Processing cascade for angiotensin peptides. Renin cleaves angiotensinogen to angiotensin-(1–10) (ANG I), which is further processed to the biologically active peptides ANG-(1–8) (ANG II) by angiotensin-converting enzyme (ACE), ANG-(1–7) by endopeptidases such as neprilysin (Nep), and ANG-(1–9) by ACE2. ANG II undergoes further processing by the carboxypeptidase ACE2 to yield ANG-(1–7) and at the amino terminus by aminopeptidase A (APA) to form ANG-(2–8) or ANG III. ANG-(1–7) is metabolized by ACE to form ANG-(1–5), and ANG III is further hydrolyzed by aminopeptidase N (APN) to yield ANG-(3–8) or ANG IV. The novel peptides ANG-(1–12) and ANG-(1–25) may be directly derived from angiotensinogen. ANG II and ANG III recognize the ANG II types 1 and 2 receptors (AT₁R and AT₂R, respectively), whereas ANG-(1–9) interacts with the AT₂R. ANG-(1–7) and Ala¹-ANG-(1–7) recognize the Mas and Mas-related G protein-coupled receptor member D (mRG-D) receptors. ANG IV recognizes the insulin-regulated aminopeptidase (IRAP). DC, aspartic acid decarboxylase. Inset: pathways for ANG-(1–7) formation that may reflect predominantly Nep or ACE2. Adapted from Chappell (29).

Fig. 2.

Influence of a high-salt (HS) diet and ovariectomy (OVX) on angiotensinogen excretion in the mRen2.Lewis rats. A: angiotensinogen excretion in female and male mRen2.Lewis under normal salt (NS, 0.5% Na) and HS (5% Na) diets for 10 wk = (5 to 15 wk of age) and in ovariectomized (OVX) females (at 5 wk of age). The mean values for angiotensinogen excretion are given above each bar. B: angiotensinogen mRNA levels and protein expression in intact and OVX females (left) and males (right) on NS or HS diets. C: urinary excretion of ANG II (top) and ANG-(1–7) (bottom) in intact or OVX females and males on NS and HS diets. Urinary levels of angiotensinogen were measured by a total angiotensinogen ELISA and angiotensin peptides by direct radioimmunoassays (RIAs); all values were normalized to urinary creatinine [mg creatinine (Cr)]. Relative angiotensinogen mRNA levels were assessed by real-time PCR and protein expression by immunoblot that was normalized to elongation factor 1- α (EF1-α). AGT, angiotensinogen; OD, optical density. *P < 0.05 vs. corresponding NS control group. Data adapted from Cohen et al. (34).

Fig. 3.

Characterization of AT₁Rs in isolated nuclei from the rat renal cortex. A: saturation binding with the antagonist ¹²⁵I-Sar¹, Thr⁸-ANG II (sarthran) in isolated renal nuclei. B: scatchard analysis of sarthran binding revealed a K_D of 0.7 nM and a B_max of 270 fmol/mg protein. C: competition studies revealed inhibition of binding with AT₁ antagonists losartan (Los), candesartan (CV), but not the AT₂ antagonist PD123319 (PD) or the AT₇/Mas receptor antagonist d-Ala⁷-ANG-(1–7) (DALA). D: cell sorting of isolated renal nuclei revealed essentially complete overlap of the nuclear marker nucleoporin 62 (Nup62) and the AT₁R. PE, phycoerythrin. E: immunoblot of renal nuclei with the AT₁R antibody revealed a single 52-kDa band. MS, molecular size. F: ANG II stimulates oxidative stress in renal nuclei measured by the increased fluorescence with the dichlorofluorescein (DCF) probe. ANG II-dependent stimulation was blocked by Los and the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI). ROS, reactive oxygen species. *P < 0.05 vs. control or ANG II-treatment. Data adapted from Pendergrass et al. (104, 105).

Fig. 4.

Characterization of the AT₇/Mas receptor in the kidney and isolated nuclei from the sheep renal cortex. A–C: immunofluorescent staining for the AT₇/Mas receptor (Alomone antibody) in proximal tubules (PT), collecting duct (CD), vasa recta (VR) and thick ascending limb (TAL) but not glomeruli (GM) or distal tubule (DT) in the sheep kidney. D–E: absence of ICC staining by preabsorption with the Mas immunogenic peptide in adjacent sections. F: single protein band in isolated nuclei from sheep cortex revealed by Alomone Mas antibody in full-length gel. G: competition for sarthran binding on isolated nuclei from sheep renal cortex. The AT₇/Mas receptor antagonist DALA and AT₁R antagonist Los exhibit complete additivity for competition of sarthran binding. In contrast, the AT₂ antagonist PD exhibits partial additivity with DALA. Mean values of percent competition are given above each bar. H: ANG-(1–7) (A7) stimulates nitric oxide (NO) release (increased DAF fluorescence) that was blocked by DALA and the nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester but not by Los or PD. *P < 0.05 vs. ANG-(1–7)-treatment. Data adapted from Gwathmey et al. (57, 58).

Fig. 5.

Characterization of ANG II and ANG-(1–7) in the mouse kidney by HPLC-RIA. HPLC/RIA analysis of pooled mouse kidney extracts from wild-type (WT; A) and tissue ACE knockout (tisACE^−/− mice; B). The HPLC fractions were measured with ANG-(1–7) (fractions 1–20) and ANG II (fractions 21–40) RIAs, respectively. Arrows indicate the elution peak times for ANG-(1–7), ANG-(3–7), ANG-(4–8), ANG II, and ANG-(2–8) or ANG III. C: reduced total ANG II content in tisACE^−/− kidney but no change in total ANG-(1–7) levels as determined by direct RIAs. D: proportion of ANG II and ANG-(1–7) and their metabolites in the mouse WT kidney from HPLC-RIA analysis. Intrarenal concentration of ANG II and ANG-(1–7) was expressed as fmol/mg protein in WT and tisACE^−/− mice. *P < 0.05 vs. WT. Adapted from Modrall et al. (88).