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Am J Physiol Heart Circ Physiol 310: H137H152, 2016.

First published October 16, 2015; doi:10.1152/ajpheart.00618.2015.

Review

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


absolute?
Mark C. Chappell
The Hypertension and Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, North
Carolina
Submitted 6 August 2015; accepted in final form 15 October 2015

Chappell MC. Biochemical evaluation of the renin-angiotensin system: the


good, bad, and absolute? Am J Physiol Heart Circ Physiol 310: H137H152, 2016.
First published October 16, 2015; doi:10.1152/ajpheart.00618.2015.The reninangiotensin 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-(17)-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.
ACE; angiotensin; heart; renin

THE RENIN-ANGIOTENSIN SYSTEM (RAS) comprises a number of


enzymatic pathways and bioactive components that underlie
diverse functional actions (Fig. 1). Although originally characterized as a circulating endocrine system that targets both
peripheral and central receptors, abundant evidence reveals a
tissue-based RAS that influences local cellular actions and
exhibits intracellular or subcellular components (2, 25, 54, 71,
74, 93, 134, 140, 151). The RAS is classically defined by the
activity of angiotensin-converting enzyme (ACE) to form angiotensin II (ANG II) and the subsequent activation of the
angiotensin type 1 receptor (AT1R) to mediate both peripheral
and central mechanisms in the regulation of blood pressure.
The ACE-ANG II-AT1R pathway is also associated with various pathologic responses including fibrosis, inflammation,
metabolic dysregulation, heart failure, cancer, aging, and diabetic injury (25, 46, 71, 93, 134). Conversely, alternative RAS
pathways may mitigate against the activation of the ACE-ANG
II-AT1R, particularly in response to the pharmacological
blockade of this axis (29, 43, 47, 119). Targeting ACE reduces
ANG II expression but enhances the ANG-(17)-AT7/MasR
axis that generally opposes the actions of the ANG II-AT1R
(29, 43, 44, 103, 119, 120). AT1R antagonists may also

Address for reprint requests and other correspondence: M. C. Chappell,


Hanes 6042, The Hypertension & Vascular Research Ctr., Wake Forest
Univ. School of Medicine, Winston-Salem, NC 27157-1032 (e-mail:
mchappel@wakehealth.edu).
http://www.ajpheart.org

increase the formation of ANG-(17) through ACE2, as well as


shunt ANG II to the AT2R pathway that shares similar properties to the ANG-(17) system (12, 25, 29, 44, 47, 119). The
RAS is composed of a complex array of components that can
be functionally partitioned into distinct receptors, peptides, and
peptidases that are downstream from the initial processing of
the precursor angiotensinogen to form ANG I by renin (Fig. 1).
The review assesses the current biochemical approaches to
quantify these components that ultimately define the status of
the RAS and to address the consistency of the expected values
of the RAS, particularly in lieu of new methodologies applied
to the quantification of endogenous angiotensin peptides in
various biological compartments including the circulation, tissues, urine, and cells.
RAS Protein Components
Angiotensinogen. A soluble glycosylated protein (molecular
mass, 50 60 kDa), angiotensinogen is the sole precursor of
angiotensin peptides and the only known substrate for renin
(Fig. 1). Circulating levels of the protein are relatively abundant (105 g or 10 g/ml; 200 nM), particularly compared with
ANG II or ANG-(17); therefore, a very small quantity (0.1
ml) of serum or plasma is required to quantify angiotensinogen
(Table 1). A specific and sensitive enzyme-linked immunoassay (ELISA) for angiotensinogen developed by Kobori and
colleagues (70) is commercially available and comprises antibodies directed against two distinct antigenic regions for the

0363-6135/16 Copyright 2016 the American Physiological Society

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Fig. 1. Processing cascade for angiotensin peptides. Renin cleaves angiotensinogen to angiotensin-(110) (ANG I), which is further processed to the
biologically active peptides ANG-(1 8) (ANG II) by angiotensin-converting
enzyme (ACE), ANG-(17) by endopeptidases such as neprilysin (Nep), and
ANG-(19) by ACE2. ANG II undergoes further processing by the carboxypeptidase ACE2 to yield ANG-(17) and at the amino terminus by aminopeptidase A (APA) to form ANG-(2 8) or ANG III. ANG-(17) is metabolized by
ACE to form ANG-(15), 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-(125) 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-(19) interacts with the AT2R. ANG-(17) and
Ala1-ANG-(17) 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-(17) formation that may reflect predominantly Nep or ACE2. Adapted
from Chappell (29).

capture and detection of angiotensinogen. The angiotensinogen


ELISA is quite sensitive (1011 g or 10 pg; 1.6 amol), exhibits
a broad detection range, and is available for multiple species
including human, rat, and mouse. There are two angiotensinogen ELISAs that detect either total (ANG I or des-ANG I) or
intact (ANG I) forms of angiotensinogen; therefore, the extent
of the renin-processed protein can be determined (IBL-Japan,
Gumma, Japan). Importantly, the angiotensinogen ELISAs are
not compatible with tissue or cell extracts as opposed to
plasma, serum, urine, and cell media, although partial purification of the tissue or cell extract may potentially remove the

interfering substances. Tissue or cellular content of angiotensinogen can be quantified by immunoblot, provided a source
of the protein is available to standardize this approach, or,
alternatively, by addition of renin to quantify ANG I generation from intact angiotensinogen that essentially comprises a
reverse renin assay.
Human urinary levels of angiotensinogen are lower than
plasma and average 10 200 ng/mg creatinine (0.2 to 4
pmol/mg Cr) (Table 1). However, elevated excretion of angiotensinogen is considered a biomarker for an activated renal
RAS in several models of renal injury that reflects the tubular
release of angiotensinogen (5, 69, 72, 113). We applied the
total angiotensinogen ELISA and immunoblot methods to
assess angiotensinogen expression in the salt-sensitive
mRen2.Lewis rat, a congenic model of an activated ACE-ANG
II-AT1R axis, and observed a marked sex difference in urinary
angiotensinogen levels (34) (Fig. 2). Male mRen2.Lewis exhibited 200-fold higher excretion of angiotensinogen than
females (2 vs. 0.01 pmol/mg Cr) (Fig. 2). Estrogen depletion
(ovariectomy) increased angiotensinogen excretion 20-fold,
whereas a high-salt (HS) diet enhanced excretion 100-fold
compared with intact mRen2 on a normal salt diet. Although
both maneuvers increase blood pressure to a similar extent,
excretion of angiotensinogen in the HS males remained sixfold
higher than the HS females (Fig. 2). Despite the marked
increases in excretion, renal mRNA and protein levels of
angiotensinogen were not increased following ovariectomy or
the HS diet, nor did we detect enhanced release of angiotensinogen from renal cortical slices in the HS mRen2.Lewis
rat (34) (Fig. 2). The elevated urinary angiotensinogen in the
mRen2.Lewis likely reflects a greater degree of filtration of the
circulating protein rather than a renal source and is perhaps a
sensitive biomarker of glomerular damage (34).
Renin. Typically regarded as the enzyme that initiates the
RAS cascade, renin is an aspartyl-type protease (30 40 kDa)
that cleaves angiotensinogen to form the inactive peptide ANG
I (Fig. 1). The enzyme is synthesized, glycosylated, and released in both inactive (prorenin) and active forms by the
juxtaglomerular cells of the renal cortex at a higher proreninto-renin ratio. A second source of renal renin is the collecting
duct cells that secrete active renin into the tubular fluid (52, 71,
82, 92, 113). There is evidence for alternative gene products of
intracellular renin in the kidney, brain, and adrenal that may

Table 1. Verified angiotensin peptide content


Compartment

ANG-(17)

Plasma, fmol/ml (22, 24, 30, 37, 78, 97100,


102, 125)
580 (580 pM)
Adrenal, fmol/g (22, 24, 30, 37, 128)
50
Kidney, fmol/g (22, 24, 37, 70, 101, 128,
140)
100200
Lung, fmol/g (22, 70)
4
Liver, fmol/g (70)

Heart, fmol/g (22, 37, 85, 94, 95, 101, 128)


530
Ovary, fmol/g (37)

Uterus, fmol/g (37)

Adipose, fmol/g (22, 23)


8
Brain, fmol/g (22, 37, 127)
212

Urine, pmol/mg creatinine (34, 45, 50, 70,


115, 132)
0.054

300400
Cells, fmol/mg (9, 38, 77, 132, 141)

ANG-(18) (ANG II) ANG-(28) (ANG III) ANG-(19) ANG-(110) (ANG I)

*Angiotensinogen (Aogen)

1 (1 pM) 10135 (10135 pM) 50,000200,00 (50200 nM)


62
50200

550 (550 pM)


3002000

110 (110 pM)


25115

150700
40240
90
10150
350
250
20150
215

12130

32
46

15

110
5

8
15

601050
560
6
1085

8
25

0.55
50250

20
85300

0.0112

Peptide values derived from radioimmunoassay (RIA)-HPLC or HPLC-mass spectrometry corrected by molecular size. Values from direct RIA or ELISA.
Tissue levels are fmol/g wet weight. Cell content is fmol/mg protein.
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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-(17) (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- (EF1-). AGT,
angiotensinogen; OD, optical density. *P
0.05 vs. corresponding NS control group.
Data adapted from Cohen et al. (34).

imply a renin-dependent pathway for the intracellular expression of angiotensin peptides (65, 76, 106, 107, 142). Apart
from its catalytic activity, prorenin may constitute an additional circulating hormone of the RAS through binding and
activation of the prorenin receptor within various tissues (52,
71, 94). A high molecular mass complex (50 60 kDa) comprised of renin and the renin binding protein N-acetyl glucosamine epimerase is typically revealed by immunoblot analysis; the complex renders renin inactive and may influence renin
secretion (64). Regarding renin measurement, the enzyme is
released by stress, dehydration, low-salt diet, RAS blockade,
and certain anesthetics (pentobarbital sodium); therefore, physiological assessment of activity should ideally be obtained by
rapid decapitation or blood withdraw by an indwelling catheter

in conscious animals. The classical method to detect renin


activity is the direct generation of ANG I from angiotensinogen
under assay conditions that protect ANG I from enzymatic
degradation and does not attenuate renin activity. The assay
requires a sensitive and quantitative method to detect the ANG
I product at 1012 g (pg) or 1015 mol (fmol) typically by
radioimmunoassay (RIA) or ELISA. To quantify the prorenin
form, samples are enzymatically treated with trypsin to release
the propeptide that occupies the active site and inhibits the
enzyme; a trypsin inhibitor (i.e., soybean trypsin inhibitor) is
then added before addition of the substrate. Prorenin activity
constitutes the difference between the total or trypsin-activated
renin and basal renin activity. Plasma contains high amounts of
angiotensinogen; thus the assessment of plasma renin activity

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reflects both active renin and endogenous ANG I-angiotensinogen in the circulation. Elased et al. (39) initially used surfaceenhanced laser deabsorption ionization (SELDI)-mass spectrometry (MS) to detect ANG I and demonstrate relative renin
activity in very low volumes of plasma samples. Camenzind et al.
(20) applied matrix-assisted laser deabsorption ionization-mass
spectroscopy (MALDI-MS) to quantify ANG I generation from
angiotensinogen with comparable renin activity values to that of
RIA methods. Renin activity is typically expressed as nanograms
or picomoles ANG I generated per milliliter of plasma per hour.
Typical plasma renin activity values range from 0.510
ngml1h1 (0.510 pmolml1h1), whereas total renin (prorenin/renin) is 3- to 10-fold higher reflecting the greater abundance of prorenin. Addition of exogenous angiotensinogen yields
maximal renin activity that is conventionally but inappropriately
termed plasma renin concentration. An alternative method is a
direct human renin assay using capture and detection antibodies
with the latter antibody directed to the active or open form of
renin. In this regard, addition of the nonpeptide inhibitor aliskerin
induces a conformation change in the enzyme that facilitates
detection of prorenin by this assay (139). Using this method, van
den Huevel et al. (139) reported mean values of active and
prorenin at 14 pg/ml (0.4 pM) and 66 pg/ml (1.9 pM), respectively, in human plasma.
Renin activity is also evident in the urine at 10% of the
plasma levels of the enzyme (84, 139). In contrast to the
circulation, there is little or no urinary prorenin, and this may
reflect the predominant luminal secretion of renin from collecting duct cells (71, 82, 84, 92). Danser and colleagues (113,
139) suggest that urinary renin may be an alternative measure
of an active RAS within the kidney. Quenched fluorescent
substrates based on the ANG-(114) sequence are available to
measure renin. Although this approach does not rely on the
detection of ANG I or an exogenous source of angiotensinogen, the sensitivity and specificity of the peptide substrates are
not comparable with angiotensinogen. A protease/peptidase
cocktail of inhibitors must be added to prevent nonspecific
cleavage of the quenched substrate, as well as addition of a
renin inhibitor such as aliskerin to demonstrate specificity of
the assay.
RAS peptidases. Peptidases are generally distinguished from
proteases by their preference to hydrolyze peptides of 5 to 30
amino acids in length. In contrast to renin, the peptidases
associated with the RAS do not exhibit exclusive specificity for
angiotensins, although certain enzymes may exhibit preferential kinetics such as ACE2 for ANG II (112, 145). An extensive
peptidase class is the metalloenzymes [ACE, ACE2, neprilysin
(Nep), aminopeptidase A] that require divalent cations for
activity; thus the assessment of activity in the circulation is
performed in serum in the absence of strong chelating agents
such as EDTA or phenanthroline.
ACE. The predominant pathway of the classical RAS is the
hydrolysis of ANG I to ANG II by the metallopeptidase ACE,
a dipeptidyl carboxypeptidase that cleaves two residues from
the COOH-terminus of peptides. The peptidase is a membranebound and glycosylated protein (120 180 kDa); however,
soluble forms of the enzyme are present in the circulation,
cerebrospinal fluid, lymph, and urine (15). ACE plays a key
role in the formation of ANG II, but the peptidase metabolizes
other peptides including bradykinin, substance P, acetyl-SerAsp-Lys-Pro, and ANG-(17) (16, 145). ACE activity is typ-

ically measured by small peptide substrates such as hippurylHis-Leu or furylacrylol-Phe-Ala-Gly-Gly in a buffer containing chloride (10 200 mM) and zinc (1100 M) for optimal
peptidase activity (24, 122). Quenched fluorescent substrates
Mca-Ser-Phe-Leu-Tyr-DNP or Mca-Tyr-Val-Ala-Arg-Ala-PheLys-DNP have also been applied for ACE measurement; however, these are not specific substrates and appropriate assay conditions that include an inhibitor cocktail to prevent nonspecific
hydrolysis by other peptidases must be considered, as well as
separate samples sets for an ACE inhibitor (i.e., captopril, lisinopril) to confirm assay specificity (81). Finally, the degree of
sample quenching of the fluorescent product should also be
assessed for each source of activity.
CHYMASE. Chymases comprise a family of serine peptidases
that may form ANG II by hydrolysis of the Phe8-His9 bond of
ANG I and pseudoprecursors [ANG-(112), ANG-(125)] (chymases) or metabolize ANG II at Tyr4-Ile5 to form ANG(1 4) and ANG-(5 8) (-chymases) (3, 27, 28, 89, 138).
Humans express -chymase, whereas rodents express primarily -chymases, as well as other isoforms (mouse mast cell
protease-4 and rat mast cell protease-5) that more closely
resemble -chymase in regard to the processing of ANG I to
ANG II (27, 28, 126). The human and mouse enzymes may
also play a role in the conversion of the endothelin precursor to
the active peptide, as well as the activation of various inflammatory cytokines (28, 126). Chymases (35 kDa) are synthesized and stored in an inactive proform within mast cells and
neutrophils that are released with other proteases (cathepsin G,
tryptases, renin) upon degranulation following injury or inflammation (28). Although chymases are soluble enzymes,
they associate with the cell membrane and may locate to the
extracellular surface of tissues following release. Fluorescentquenched substrates for chymase include succinyl-Ala-AlaPro-Phe-amido-4-methylcoumarin and succinyl-Leu-LeuVal-Tyr-amido-4-methylcoumarin. Addition of the serine
protease inhibitor chymostatin is typically used to demonstrate specificity; however, chymostatin inhibits other ANG
II-generating enzymes (cathepsin G, elastase-2) and more
selective chymase inhibitors should be considered (116
118, 123, 146). The extent that chymase or other peptidases
participate in the formation of circulating or tissue ANG II
through an ACE-independent pathway remains an area of
significant debate (21).
ENDOPEPTIDASES. Nep (95 kDa) and other endopeptidases
(thimet oligopeptidase, 80 kDa; and prolyl oligopeptidase, 76
kDa) process ANG I directly to ANG-(17) without the requisite formation of ANG II (Fig. 1). These peptidases hydrolyze interior bonds of the peptide and prefer aromatic and/or
hydrophobic residues, but prolyl oligopeptidase specifically
cleaves the COOH-terminus of proline and hydrolyzes the
Pro7-Phe8 bond of both ANG I and ANG II to form ANG-(17)
(29, 112, 145). Circulating forms of soluble Nep are present in
the serum, urine, and cerebrospinal fluid (111, 129). Following
chronic ACE inhibition, circulating levels of ANG-(17) are
markedly higher that reflect processing of ANG I by vascular
Nep, as well as the reduced metabolism by ACE (Fig. 1) (29).
Various quenched substrates are available to measure Nep and
other endopeptidases (81). The quenched peptide MCA-ArgPro-Pro-Gly-Phe-Ser-Ala-Phe-Lys-DNP may be an appropriate
dual substrate for ACE and Nep (M. C. Chappell, unpublished
observations). Again, the assay conditions require inhibitors to

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ASSESSMENT OF THE RAS

prevent nonselective hydrolysis of the substrate, as well the


addition of a Nep inhibitor (thiorphan, SCH39370) to demonstrate
specificity. Miners et al. (86) immunoprecipitated Nep before the
addition of the fluorescent substrate, and this maneuver may
enhance the specificity of the assay. This approach should be
applicable to other peptidases provided the appropriate antibodies
are available to pull down the enzyme and do not inhibit the active
site.
ACE2. ACE2 is a membrane-bound monocarboxypeptidase
(120 kDa) that converts ANG II directly to ANG-(17). Circulating levels of ACE2 are typically quite low particularly
compared with ACE; however, serum ACE2 activity is elevated in diabetes, heart failure, and hypertension (41, 136,
148). ACE2 has a potentially significant role in the RAS
pathway as a single catalytic step degrades ANG II but activates the ANG-(17) axis (Fig. 1) (29). ACE2 assays use the
quenched fluorescent substrates Mca-Ala-Pro-Lys-DNP or
Mca-Tyr-Tyr-Val-Ala-Asp-Pro-Lys-Dnp (111). These substrates are not specific for ACE2, and addition of other inhibitors to block ACE, Nep and prolyl oligopeptidase, are critical
to demonstrate assay specificity, as well as additional samples
that include a specific ACE2 inhibitor (MLN4760, DX600).
These fluorescent assays can be quantified for enzyme
concentration given a standardized source of each peptidase
is available (R&D Systems, Minneapolis MN). Using this
approach, Rice et al. (111) reported the concentration of
ACE in human serum averaged 7 nM in over 500 subjects,
whereas ACE2 content was 200-fold lower (33 pM) and
detectable in a minor fraction (10%) of this population.
Circulating Nep content (290 pM) was also lower than ACE
and evident in 30% of these patients (111).
Aminopeptidases. The role of aminopeptidases in the RAS is
generally considered a degradative pathway that initiates the
metabolism of ANG II (Fig. 1). Recent studies suggest that the
NH2-terminal processing of ANG II to ANG-(2 8) or ANG III
by aminopeptidase A may yield a peptide selective for brain
AT1 receptors and kidney AT2 receptors (18, 25, 68, 103, 109).
Further NH2-terminal processing of ANG III to ANG-(3 8) or
ANG IV yields another bioactive peptide that interacts with the
insulin-regulated aminopeptidase (4). Fluorescent substrates
are available for aminopeptidases A and N as well as general
(amastatin, bestatin) and more selective inhibitors to distinguish these enzymes (18).
Peptidase assays. Endogenous peptide substrates including
ANG I, ANG II and ANG-(112) can be directly used to
quantify peptidase activities (79). One advantage is that the
contribution of various peptidases for a given peptide can be
directly compared to determine the predominant activity in a
particular tissue or treatment. In contrast, peptidase activities
derived by different synthetic substrates are not directly comparable unless standardized to enzyme content. Moreover, use
of endogenous peptides may reveal novel peptidase activities
involved in angiotensin processing (62, 144). Peptidase assays
developed in the authors laboratory use 125I-radiolabled peptides coupled to HPLC-based separation and in-line -detection (129). Typically, microliter amounts of serum or microgram quantities of tissue are required with this approach. We
find that circulating ACE and ACE2 activities were significantly increased in a model of diabetic hypertension; however,
ACE activity remained far higher than ACE2 which may
contribute to the greater content of ANG II in diabetic

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mRen2.Lewis rats (148). Kinetic analysis using both labeled


and unlabeled ANG II confirmed a sevenfold increase in the
Vmax value for serum ACE2 in the female diabetic rat (148).
Angiotensin processing in tissues, cells, and plasma was
recently assessed by HPLC-MS. Hildesbrand et al. (62) used a
HPLC-tandem quadropole system (HPLC-MS/MS) to reveal
metabolism pathways from ANG I to its NH2-terminal metabolites ANG-(510) and ANG-(4 10), as well as ANG II and
ANG-(17) in immobilized proteins from plasma. Suski et al.
(133) find that ANG I was primarily converted to ANG-(17)
in vascular smooth muscle cells (VSMC) by HPLC-MS/MS
and support an earlier study (32) that thimet oligopeptidase
directly processed ANG I to ANG-(17) in rat VSMCs. Finally, Grobe and colleagues (6, 53) applied in situ MALDI to
characterize both renal and cardiac metabolism of exogenous
ANG II. In this approach, ANG II was incubated on the tissue
surface and the MS matrix subsequently layered over each
tissue section. The matrix was subjected to MALDI-MS analysis, and the metabolism products were identified and localized
to distinct areas of the kidney. ANG-(17) was the primary
product from ANG II in the renal cortex, whereas ANG III was
the major metabolite in the medulla (53). In the heart, ANG III
and ANG-(17) were major products of ANG II metabolism,
catalyzed by aminopeptidase A and ACE2, respectively (6).
These data confirm earlier HPLC-based studies on the contribution of ACE2 to ANG-(17) formation in the mouse and
human heart (49, 152). Caveats with this approach are that
MALDI-MS cannot distinguish intracellular versus membrane
or extracellular processing and requires relatively high substrate concentrations to detect peptidase activity that may not
reflect endogenous processing pathways. However, given the
rapid advancement of this technology, future systems will
likely develop the required sensitivity and resolution to appropriately detect peptides in situ, as well as characterize the
extent of enzymatic processing.
Receptors. ANG II and ANG III interact with two G proteincoupled receptors identified as the AT1 and AT2 subtype,
whereas ANG-(17) and [Ala1]-ANG-(17) recognize the Mas
and Mas-related G protein-coupled receptor member D
(mRG-D) receptors, respectively (12). Immunoblot techniques
can provide a relative quantification of protein expression, as
well as tissue and cellular distribution; however, the commercially obtained AT1 and AT2 receptor antibodies are notorious
for their nonspecific nature (59, 61). Receptor antibody specificity is complicated by 1) posttranslational processing events
such as glycosylation or proteolysis that may yield unexpected
molecular size bands for the receptor in different tissues or
species, 2) the very low level of receptor expression particularly compared with other RAS proteins, and 3) the lack of
appropriate controls for each antibody. The sole reliance on
antibody-based immunoblot methods to assess receptor expression underlies the need to standardize the receptor antibodies to
ensure some degree of reproducibility among laboratories.
Issues with antibody specificity are certainly not limited to
the RAS components and present a major concern regarding
the unreliability of antibodies against their intended targets
(13). Indeed, the National Institutes of Health has recently
promoted a more rigorous approach to ensure reliability and
overall reproducibility in preclinical experimental studies (http://
www.nih.gov/science/reproducibility/). Uses of knockout transgenic mice, knockdown approaches in cells, or overexpression of

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the target protein are appropriate controls to test antibody specificity for immunoblot and immunocytochemistry methods, although these approaches are not directly comparable with other
species or different tissues. Moreover, knockdown of the functional domain does not ensure that the remaining protein will not
be recognized by the antibody. Blockade of the immunoreactive
signal by the immunizing peptide or full-length protein is an
additional approach to address specificity of the immunoblot or
immunocytochemistry signal provided a pure source of the peptide/protein is available; however, antigenic sites exposed by SDS
denaturation, formalin fixation, or antigen retrieval may not be
accessible for proteins in solution.
The characterization of angiotensin receptors is further
complicated by the fact that these proteins do not reside or
exclusively localize to the plasma membrane. In lieu of
earlier studies on intracellular peptide receptors, ANG II
and ANG-(17) receptors were characterized on isolated
nuclei from rat and sheep kidney (54). In rat nuclei, binding
studies with the nonselective antagonist 125I-[sarcosine1,
threonine8]-ANG II (sarthran) and competition with selective antagonists revealed exclusive expression of the AT1
receptor (104, 105) (Fig. 3, AC). Fluorescence-activated
cell sorting demonstrated essentially complete overlap of
the fluorescent signature for the AT1 receptor and the
nuclear marker nucleoporin; the immunoblot confirmed a
single protein band for the renal nuclei with the same AT1R

antibody (Fig. 3, DE). Functional studies revealed that


ANG II stimulated reactive oxygen species and was blocked
by both an AT1R antagonist and a NAD(P)H inhibitor (Fig.
3F). A similar approach was taken for the AT7/Mas receptor
in isolated nuclei of the sheep kidney (5558). An alomone
antibody (Alomone Labs, Tel Aviv, Israel) revealed Mas
receptor expression along the tubular elements of the kidney
that was abolished by antibody preabsorption with the
immunogenic peptide; a single band (35 kDa) was evident
on the full-length immunoblot of isolated nuclei (Fig. 4,
AF). Competition of sarthran binding in nuclei revealed
complete additivity with the AT1R antagonist losartan and
the AT7R antagonist [D-Ala7]-ANG-(17) (A779), but partial additivity with the AT2R antagonist PD123319 suggesting the AT7R and AT2R antagonists are not entirely selective for the sarthran ligand (Fig. 4G). However, only the
[D-Ala7]-ANG-(17) antagonist and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester abolished
the ANG-(17)-stimulated release of nitric oxide on renal
nuclei as detected by diaminofluorescein fluorescence (Fig.
4H). In regard to the AT2R, Padia et al. (103) find that the
receptor protein resides primarily in the cytosolic compartment and rapidly inserts into the apical membrane of the
proximal tubules following dopaminergic stimulation to facilitate a natriuretic response. Interestingly, translocation of the
AT2R in spontaneously hypertensive rats was blunted com-

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 KD of 0.7 nM and a Bmax 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-(17) (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).
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Fig. 4. Characterization of the AT7/Mas receptor in the kidney and isolated nuclei from
the sheep renal cortex. AC: 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. DE: 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-(17)
(A7) stimulates nitric oxide (NO) release
(increased DAF fluorescence) that was
blocked by DALA and the nitric oxide
synthase inhibitor NG-nitro-L-arginine
methyl ester but not by Los or PD. *P
0.05 vs. ANG-(17)-treatment. Data
adapted from Gwathmey et al. (57, 58).

pared with normotensive Wistar-Kyoto rats (103). Biotinylation studies combined with an AT2R antibody was used to
reveal localization of the receptor on the plasma membrane
(103). It is not currently known whether the other angiotensin
receptor subtypes exhibit a similar type of regulation. In this
regard, a careful assessment of the subcellular fractions or
intracellular organelle for angiotensin receptor expression is
warranted. Moreover, these and other studies support the intracellular expression of G protein-coupled receptors within the
kidney and other tissues, as well as emphasize the necessity for
multiple approaches in the identification and characterization
of angiotensin receptors (17, 35, 40, 74, 132, 151).
RAS Peptides
Interest in angiotensins other than ANG II and ANG III was
stimulated by identification of endogenous ANG-(17) over 25
years ago (30), as well as subsequent evidence for an ANG(17) receptor (121) and a converting enzyme (137). [Ala1]ANG II and [Ala1]-ANG-(17) were recently identified in
human plasma, likely arising from decarboxylation of the

NH2-terminal aspartic acid residue (67, 75). Functionally,


[Ala1]-ANG II may not distinguish AT1 or AT2 receptors (67,
149), whereas [Ala1]-ANG-(17) recognizes the Mas-related
receptor mRG-D to induce vasorelaxation (12, 75). Both
[D-Pro7]-ANG-(17) and the AT2 antagonist PD123319
blocked the actions of [Ala1]-ANG-(17), but not [D-Ala7]ANG-(17) (75). Jankowski et al. (66) identified [Pro1,
Glu2]-ANG II in human plasma and this peptide exhibits a
greater affinity for the Mas receptor than ANG-(17); however, the pathway for the peptides formation is unknown. The
ANG II metabolite ANG-(3 8) or ANG IV has distinct functional properties through the interaction with the insulin-regulated aminopeptidase, although the peptide also stimulates the
AT1R (4, 80, 150). ANG-(19), a potential product of ACE2
processing of ANG I, appears to functionally interact with the
AT2 receptor (101). Finally, Nagata and colleagues (89, 90)
identified the intermediate precursors ANG-(112) in rat and
ANG-(125) in human urine. ANG-(112) is processed by
ACE and chymase to ANG II, and Nep directly generates
ANG-(17), whereas ANG-(125) is converted by a chymase-

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like enzyme to ANG II (3, 89, 143). The circulating and tissue
pathways for the generation of ANG-(112) and ANG-(125)
are not currently known but are likely renin independent.
The accurate assessment and identification of angiotensins
are critical to ascertain the status of the RAS, particularly in
pathological conditions or following therapeutic intervention;
however, their evaluation is hampered by the extremely low
level of peptide expression in the fentomole per gram or
fentomole per milliliter range, interfering substances in samples that may be present at a far higher concentration, susceptibility to metabolism in sample collection and processing, the
peptides shared sequence, and the specificity of detection. All
these factors may contribute to the marked variability in
angiotensin values evident in the literature and raise concerns
on the identity of ANG II or other angiotensins detected in
tissues and the circulation, as well as the extent that altered
peptide content truly reflects or contributes to a particular
phenotype or treatment response.
RIA/ELISA. RIA and ELISA are invaluable assays to characterize angiotensins, and they remain the most used biochemical methods to quantify endogenous peptides. Under optimal
assay and extraction conditions, both methods should allow for
the sensitive and relatively specific detection of ANG II and
other angiotensins at fentomole (pg) quantities in plasma,
tissue, urine, and cells. Since angiotensins share an identical
NH2-terminus except for the Ala1-forms of ANG II and ANG(17), antibodies for ANG II, ANG-(17), ANG-(19), ANG I,
and ANG-(112) are directed against their unique COOHterminus. The ANG II and ANG-(17) antibodies, for example,
typically distinguish a single amino acid difference (Phe8)
between ANG II and ANG-(17). Importantly, these antibodies
are not selective against the NH2-terminal portion of the
peptide (30). Thus an ANG II RIA distinguishes ANG-(17)
and ANG I but will recognize ANG III, ANG IV, and ANG(4 8) (37). Similarly, an ANG-(17) antibody recognizes the
NH2-terminal metabolites ANG-(27) and ANG-(37), but not
ANG-(4 7), ANG II, or ANG I (30). These assays can be used
across species as the COOH-terminal portion is conserved for
ANG-(17), ANG II, and ANG I; however, larger peptides
such as ANG-(112) or ANG-(125) exhibit different COOHterminal residues that are species specific and require unique
antibodies for detection (89, 90). Apart from their nonselectivity for NH2-terminal metabolites of angiotensin peptides,
RIA or ELISA may detect a significant degree of nonspecific or
background immunoreactivity that reflects the sample source
and extent of purification or extraction, as well as the assay
antibodies (37). Therefore, chromatographic separation should
be routinely coupled to RIA or ELISA to verify the identity of
immunoreactive peptides, as well as account for nonspecific
material in plasma or tissue samples. Peptide fractionation by
HPLC provides this additional level of discrimination based on
the resolution and high recovery of the sample peptides, rapid
separation time, and general compatibility with RIA methods
(22, 30, 37, 78, 99). In addition, the different characteristics for
each antibody-based assay must be appreciated, particularly
those obtained from commercial sources. The stated crossreactivity of the Peninsula human ANG-(112) ELISA for
ANG I is 0%; however, the Peninsula rat/mouse ANG-(112)
ELISA cross-reacts 60% with ANG I (Peninsula Lab Int, San
Carlos, CA). Thus HPLC fractionation would be essential to

distinguish endogenous ANG I and ANG-(112) content in the


rodent with the Peninsula ELISA.
We previously reported that renal ANG II content declined
80% in mice that lack membrane ACE, a transgenic model
developed by Bernstein and colleagues (88) that expresses
soluble but not the tissue-bound enzyme. ANG II content was
quantified directly by a commercial ANG II RIA (ALPCO
Diagonostics, Salem, NH) that recognizes ANG III, ANG IV,
and ANG-(4 8), whereas ANG-(17) was detected by a custom RIA that also detects ANG-(27) and ANG-(37) (30).
Renal tissue was extensively extracted with cold HCl-ethanol,
proteins precipitated by heptafluorobutyric acid (HFBA) and
peptides purified on C18 SepPak columns (30, 88). To confirm
ANG II identity, pooled renal extracts were fractionated by
HPLC using the HFBA system developed in the authors
laboratory that achieves baseline separation of immunoreactive
angiotensins, and the fractions were assayed by separate RIAs
(30). ANG II was the primary immunoreactive peak in both the
wild-type (WT) and transgenic ACE/ mice, but the ANG II
peak was markedly lower in the ACE-deficient mice (Fig. 5).
Note that ANG III and ANG-(4 8) represent minor immunoreactive peaks that are essentially absent in the transgenic
kidney. Renal ANG-(17) content was unchanged despite the
marked reduction in ANG II and suggests that a non-ACE2
pathway contributes or maintains tissue ANG-(17) (Fig. 5).
Overall, this approach confirmed that the two RIAs identified
predominantly NH2-terminally intact ANG II and ANG-(17)
in the mouse kidney, as well as demonstrate the greater ratio of
ANG II to ANG-(17) in the WT kidney. However, these
results do not ensure that ANG II or ANG-(17) RIAs primarily detect both peptides in all experimental situations and
verification of the direct assay values is essential for each tissue
source and species.
Campbell and colleagues (2224, 78) developed an alternative approach with RIAs specific to the NH2-terminus of intact
and smaller metabolites rather than the COOH-terminus of
angiotensins. To distinguish peptides that differ in their
COOH-terminal sequence, samples are extracted in guanidine
thiocynate, acetylated, subjected to HPLC separation, and the
fractions analyzed by the different NH2-terminally-specific
RIAs. An advantage of the approach is that all peptides that
share the identical NH2-terminus [i.e., ANG I, ANG II, and
ANG-(17)] are assayed by one RIA antibody rather than
multiple assays, and all reported values reflect HPLC fractionation. Disadvantages of the HPLC-RIA approach are the required equipment, expertise, and the lack of an automated
method for analysis and that HPLC separation generates a
significantly larger sample size for RIA or ELISA quantification. Moreover, the NH2-terminal RIAs are not commercially
available and cannot be applied for direct peptide measurements. Finally, it is not clear whether HPLC-RIA can resolve
[Ala1]-isoforms or other posttranslational modifications of
ANG II and ANG-(17).
LC-MS/MS. RIA and ELISA exhibit the requisite sensitivity
and specificity to detect endogenous angiotensins; however,
these methods are highly dependent on the characteristics for
each antibody, as well as the optimal sample extraction and
assay conditions. Angiotensin RIAs or ELISAs are typically
directed against the unique COOH-terminus of the peptide, and
they fail to distinguish the NH2-terminal metabolites unless
coupled to HPLC. LC-MS/MS directly detects peptides based

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Fig. 5. Characterization of ANG II and


ANG-(17) in the mouse kidney by HPLCRIA. 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-(17) (fractions 120) and ANG II
(fractions 21 40) RIAs, respectively. Arrows indicate the elution peak times for
ANG-(17), ANG-(37), 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-(17) levels as
determined by direct RIAs. D: proportion of
ANG II and ANG-(17) and their metabolites in the mouse WT kidney from HPLCRIA analysis. Intrarenal concentration of
ANG II and ANG-(17) was expressed as
fmol/mg protein in WT and tisACE/
mice. *P 0.05 vs. WT. Adapted from
Modrall et al. (88).

on their unique mass to charge spectra and exhibits the capability for quantification of endogenous peptides (83). Unlike
the antibody-based RIA or ELISA, MS can potentially resolve
posttranslational events such as peptide phosphorylation, amidation, acetylation, decarboxylation, or other peptide forms.
Extracted samples are initially resolved by a chromatographic
step [HPLC, ultrahigh-pressure LC (UPLC), or nano-LC], the
eluent volatized, and the peptide or resultant fragment ions
detected by MS [single or tandem, time of flight (TOF), ion
trap, or their combination]. A key issue with MS is that a
number of components are detected following the LC separation and may render very complex spectra with similar massto-charge ratios, particularly as the prior chromatographic step
does not resolve all individual peptides, and endogenous peptides are typically 103-109 less abundant than other species in
plasma and tissues (11, 83). To address this issue, Schulz et al.
(125) developed a hybrid approach to selectively enrich ANG
II from plasma extracts by absorption to an ANG II antibody
before quantification by HPLC-MS/MS. These investigators
quantified ANG II at 20 fmol/ml (20 pM) in human plasma
that is comparable wth RIA or HPLC-RIA values, although
other NH2-terminal metabolites of ANG II were not reported
(125) (Table 1). Addition of isotopically labeled ANG II is key
in the MS approach to account for extensive sample handling
and extraction, as well as the extent of peptide ionization.
Poglitsch and colleagues (60, 108) have recently developed a
fingerprint HPLC-MS/MS approach to quantify 10 angiotensins in plasma with a comparable sensitivity to RIA; however, the addition of exogenous renin is apparently required for
peptide measurements in human plasma. HPLC-MS/MS may
potentially become the standard method to quantify angiotensins, although the complexity, expertise, and cost required

for these systems are limiting factors for most laboratories at


the current time. The fingerprint MS is available as a fee for
service to quantify angiotensins in plasma and tissues (Attoquant, Vienna, Austria).
Other approaches. Additional methods to quantify angiotensin peptides in plasma and tissues include direct HPLC and
capillary electrophoresis. Both methods are based on the
direct determination of angiotensins by ultraviolet absorption (UV, 180 220 nm) following an initial chromatographic or electrophoretic separation. The concerns here are
that neither approach achieves complete separation of all
peptides (angiotensin and non-angiotensin), and UV detection does not discriminate among the peptide milieu in
contrast to RIA, ELISA, or MS methods. Therefore, a UV
peak may be comprised of multiple species, and alterations
in peak area between experimental groups cannot be readily
attributed to one peptide. Moreover, the reported sensitivity
or limit of quantitation of 3 pmol for the HPLC analysis is
not adequate to detect authentic levels of endogenous peptides (16). ANG II and ANG-(17) values derived from
these methods are typically far higher in plasma (26, 33, 51,
135), kidney (33, 110, 114), vasculature (48), adipose tissue
(33, 115), and heart (42) than by RIA, HPLC-RIA, or
HPLC-MS methods (Table 1). Indeed, two recent studies
quantified mouse renal ANG-(17) at 1,000,000 fmol/g
tissue (110) and rat plasma ANG II at 300,000 pM by
HPLC analysis alone (33). Rubio-Ruiz et al. (115) reported
an ANG-(17) concentration of 6,000 pM in serum and
500,000 fmol/g in adipose tissue from male Wistar rats by
capillary electrophoresis.
Sample handling and extraction. Appropriate handling and
subsequent extraction of plasma and tissue samples cannot be

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overly emphasized for the accurate measurement of endogenous angiotensins. Regardless of the detection method by RIA,
HPLC-RIA, or HPLC-MS, improper sampling handling and
extraction may yield artifactually high or low peptide values
that do not reflect endogenous content. The sensitivity required
to quantify endogenous angiotensins also increases the potential for artifacts or interfering substances whether using RIA or
MS approaches. In general, animals should be maintained in a
calm environment and anesthetic agents such as pentobarbital
sodium that markedly activate the RAS avoided. Blood samples are collected directly into an inhibitor cocktail to block
renin and other peptidases that process or metabolize angiotensin peptides; the obtained plasma should be subsequently
stored at 80C or immediately extracted. Tissue samples
should be rinsed of blood and rapidly frozen in liquid nitrogen
or an ethanol-dry ice bath or immediately extracted. Pepstatin,
an aspartyl protease inhibitor, may not be sufficient to completely block renin activity, and a more specific renin inhibitor
should be considered (73, 98). Moreover, the typical inhibitor
cocktails for protein immunoblot analysis may be inadequate to
attenuate angiotensin processing of samples in physiological
buffers.
Various extraction methods that rapidly abolish peptide
metabolism, deplete the sample of contaminating proteins and
other substances that may interfere with the assay and enrich
peptide content in a manageable volume include acid (HCl)ethanol, pure methanol or acetonitrile, guanidine thiocynate,
and immediate immersion in a boiling water bath with subsequent acidification and tissue homogenization (22, 24, 30, 37,
90, 130). The precipitated proteins are removed by high-speed/
ultracentrifugation or molecular cut-off filters, and the supernatant is applied to solid-phase extraction columns (C18 or
phenyl). Both angiotensin and nonangiotensin peptides are
absorbed to these columns, whereas proteins and other substances are not retained; the peptides are subsequently eluted in
an organic solvent (methanol or acetonitrile). Peptides standards (i.e., 125I-ANG II, ANG II, or isotopically labeled ANG
II) should be routinely added to determine overall recovery in
the extraction procedure for quantification methods, and particularly to account for matrix effects on MS ionization that
may vary among samples or MS analysis runs. Recovery for
plasma samples typically ranges from 70 to 90%, whereas
tissues are generally lower in the 40 70% range. Appropriate
blanks must be included that contain all components of the
extraction method (apart from the actual sample) and assayed
in parallel with samples.
Angiotensin values. The range of angiotensin values for
plasma and tissues in Table 1 reflects studies using HPLC-RIA
or HPLC-MS quantification, as well as consideration of the
appropriate sample collection and extraction methods (2224,
37, 85, 97100, 127, 128). In lieu of these approaches, the
NH2-terminally intact forms of ANG II, ANG-(17), and ANG
I are the predominant peptides identified in plasma and various
tissues. Quantitation of the cell and urinary data reflects direct
RIA- or ELISA-based methods (9, 34, 38, 45, 50, 70, 77, 115,
131, 133, 139, 141, 148).
CIRCULATION. ANG II values in human, mouse, and rat
plasma are quite low and average 550 fmol/ml (550 pM)
(Table 1). Human plasma levels of the ANG II isoforms
[Ala1]-ANG II and [Pro1, Glu2]-ANG II average 6 and 4 pM,
respectively, as quantified by MALDI (66, 67). Circulating

ANG-(17) values exhibit a similar range to ANG II, although


the peptide is markedly increased following ACE inhibitor
treatment, whereas endogenous levels of the ANG-(17) isoform [Ala1]-ANG-(17) have not been quantified (Table 1).
ANG I values are somewhat higher than ANG II, provided
renin activity is attenuated during the sample collection and
processing, whereas ANG-(19) levels are very low (Table 1).
Adequate plasma volumes to detect these peptides in this range
require 3 to 5 ml of extracted plasma (4 10 ml blood) to assay.
In regard to the MS analysis of circulating angiotensins, the
peptide profile reported by Poglitsch and colleagues (60, 108)
is difficult to interpret as addition of exogenous renin to human
plasma samples is apparently necessary to quantify circulating
angiotensin peptides. This approach does not reflect the actual
angiotensin content in the circulation as renin addition fails to
account for the contribution of membrane-bound peptidases
(i.e., Nep, ACE2, ACE, APA) nor the vascular release of
angiotensin peptides (31, 63, 87). Wysocki et al. (147) recently
evaluated plasma angiotensins from male WT mice by the
fingerprint MS method without the addition of renin. Surprisingly, plasma levels of ANG III greatly exceeded ANG II
(1,130 vs. 7 pM), whereas similar levels of ANG I and
ANG-(210) (1,000 and 700 pM) were reported (147). Little
detail was provided on the extraction method or inhibitors
employed, and the high plasma content of ANG III and
ANG-(210) may reflect inadequate blockade of aminopeptidase activity particularly given the use of the anesthetic pentobarbital (147). Indeed, their additional analysis of plasma
peptides from female WT mice anesthetized with ketamine
revealed far lower plasma ANG III, although the levels of
ANG III were twofold higher than ANG II (160 vs. 74 pM)
(147). Olkowicz et al. (102) quantified plasma angiotensin by
MS in male WT mice and in apolipoprotein E/ mice that
exhibit a stimulated RAS. Plasma levels of ANG II, ANG III,
and ANG-(17) were 36, 32, and 63 pM, respectively; however, ANG IV (158 pM) was the predominant peptide in WT
mice (102). ANG II and its COOH-terminal intact metabolites
were elevated 3- to 10-fold in the apolipoprotein E/ mice,
but ANG IV remained the major peptide (550 pM); plasma
levels of ANG-(17) were unchanged (102). Although the
ANG II and ANG-(17) values were comparable with RIAbased studies, the higher ANG III and ANG IV content may
again reflect inadequate inhibition of aminopeptidases by
addition of a standard protease inhibitor cocktail in contrast
to extraction methods specifically designed to inhibit peptide processing (102). Finally, Ali et al. (7) quantified
plasma ANG II and ANG-(17) content at 20,000 and
40,000 pM, respectively, in WT mice using UPLC-quardruple (Q)TOF. The presence of other ANG II or ANG(17) metabolites were not reported, but the extreme ANG
II and ANG-(17) values by this method likely reflect
inadequate inhibition of plasma peptidases during blood
collection and extraction (7).
Tissues. Tissue expression of angiotensins is widespread;
however, there are marked differences in the tissue content of
these peptides (Table 1). The adrenal typically expresses the
highest content of ANG II from 300 to 2,000 fmol/g tissue with
far lower levels of ANG III and ANG-(17) (Table 1). ANG II
content in other peripheral tissues including the kidney, lung,
and liver average of 100 to 700 fmol/g (Table 1). Within the rat
kidney, ANG II, ANG-(17), and ANG I levels were higher in

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the medulla than the cortex (direct RIAs) (91, 104); Pendergrass et al. (104) confirmed the predominant ANG II identify in
both cortex and medulla by HPLC-RIA. In comparison, significant levels of ANG II (300 fmol/g tissue) were quantified
in the ovary and uterus by Senanayake and colleagues (37).
Apart from these tissues, central and peripheral tissues including the heart and adipose express lower and somewhat more
variable ANG II levels of 2150 fmol/g tissue (Table 1).
In regards to MS analysis, few studies have quantified ANG
II and ANG-(17) in tissue. Ali et al. (7, 8) reported ANG II
and ANG-(17) levels of 200,000 and 350,000 fmol/g in
mouse kidney and 25,000 to 60,000 fmol/g tissue in rat kidney
cortex by UPLC-QTOF (7, 8). They also quantified ANG II
and ANG-(17) content in mouse adipose tissue at 150,000 and
350,000 fmol/g tissue, respectively (7). It is difficult to reconcile that the kidney and adipose exhibit the same synthetic
capacity to generate essentially identical levels of ANG II or
ANG-(17). Moreover, extraction of tissue with a very high
protease content such as the kidney in a Tris lysis buffer at
neutral pH is likely insufficient to abolish processing and may
contribute to the high peptide content reported for kidney (7,
8). The stated limit of quantitation for the UPLC-QTOF for
ANG II (500 fmol) and ANG-(17) (800 fmol) also does not
appear adequate to quantify the authentic peptide content in
plasma and tissues (7). In contrast to the Ali studies, Wysocki
et al. (147) quantified far lower levels of ANG II and ANG(17) (293 and 95 fmol/g tissue, respectively) in the mouse
kidney by fingerprint MS; these values are clearly more consistent with the literature, although the ANG III content (185
fmol/mg tissue) was relatively high at 60% of the total ANG II
(Fig. 5, Table 1). Finally, Burger et al. (19) found a higher
ANG II-to-ANG III ratio (700 vs. 130 fmol/g tissue) in mouse
kidney using guanidine extraction and MS. In this case, guanidine may attenuate aminopeptidase and other peptidase activities more effectively to prevent the artifactual generation of
peptides during tissue extraction.
INTERSTIAL CONTENT. The extracellular or interstial content of
ANG II in heart and kidney was assessed by direct RIA and
HPLC-RIA. These studies use an implanted dialysis probe
permeable to low molecular substances that sample the local
release or formation of angiotensin peptides in the interstial
space. DellItalia and colleagues (14, 36) reported interstial
ANG II levels in the canine heart at 0.6 to 5 pmol/ml (0.6 5
nM) by HPLC-RIA. Intravenous infusion of ANG I alone or
combined with an ACE inhibitor did not impact the interstial
ANG II levels. Schuijt et al. (124) found low to undetectable
levels of ANG II (30 fmol/ml or 30 pM) in interstial fluid of
the ovine heart also quantified by HPLC-RIA. In the rat kidney,
Kemp et al. (68) report comparable ANG II and ANG III
interstial content (80 fmol/ml or 80 pM) using the HFBAHPLC system to separate the two peptides before RIA detection. This study finds a higher ANG III-to-ANG II ratio than
that in the circulation or tissues, which may reflect the significant level of ectocellular APA activity in the renal tubules and
suggests significant angiotensin processing in the interstial
space. Finally, Nishiyama et al. (95, 96) reported higher renal
interstial levels of ANG II and ANG I (13 nM) by direct
RIAs. The interstial ANG II levels were not responsive to
peripheral ACE inhibition or volume expansion, further suggesting a local mechanism of peptide expression distinct from

H147

the circulation, albeit the identity of ANG II or ANG I


immunoreactivity was not established in these studies (95, 96).
URINE. The urinary components of the RAS are of interest
regarding their potential application as biomarkers of renal
injury or RAS activation (5, 72, 113). Although excretion of
the RAS proteins including angiotensinogen, renin, and ACE2
are more routinely assessed, the urinary levels of ANG II and
ANG-(17) were quantified by direct RIA or ELISA (Table 1).
Urine contains multiple peptidase activities, and the samples
should be collected under conditions that limit ex vivo metabolism (acidification, addition of inhibitors, or collection on dry
ice) (50). Chan and colleagues (131) reported higher ANG II
excretion in the diabetic Akita mice compared with WT (25
vs. 2 pmol/mg Cr), whereas urinary ANG-(17) levels were
50% lower in the diabetic mice (3 vs. 7 pmol/mg Cr),
suggesting an imbalance of ANG II to ANG-(17) in the
diabetic kidney; these peptides were quantified by Bachem
ELISAs (Torrance, CA). Gilliam-Davis et al. (50) quantified
ANG II (0.4 pmol/mg Cr) and ANG-(17) (0.2 pmol/mg Cr) in
adult male Fisher 344 rats by direct RIAs. Both ANG II and
ANG-(17) excretion increased twofold in aged Fisher rats,
suggesting activation of the renal RAS in this model of aging.
Chronic treatment with an AT1R antagonist lowered the excretion of both peptides despite a marked increase in circulating angiotensins and further distinguishes compartmentalization of the renal and circulating RAS (50). Urinary levels of
both ANG II and ANG-(17) were elevated in female and male
mRen2.Lewis on the HS diet; however, ovariectomy of the
hypertensive females did not increase peptide excretion despite
a 20-fold increase in urinary angiotensinogen (Fig. 2) (34).
Finally, ANG-(17) excretion was significantly lower in a
cohort of hypertensive patients compared with the normotensive group (0.05 vs. 0.11 pmol/mg Cr) using a custom RIA to
quantify ANG-(17); ANG-(17) identity in human urine was
verified by HPLC-RIA (45).
CELL MODELS. In an extensive analysis of angiotensin regulation in rat VSMCs using direct RIAs (Peninsula), Lavrentyev
et al. (77) reported that ANG II content increased threefold
(50 to 150 fmol/mg protein) in response to high glucose
conditions. ANG II content also increased in the cell media
with high glucose (100 to190 fmol/ml). In contrast, basal
ANG-(17) (260 fmol/mg protein) was higher than ANG II,
and ANG-(17) content was reduced fivefold under hyperglycemic conditions that was also associated with a reduction in
ACE2 (77). In a human mesangial cell line, Boim and colleagues (141) reported comparable ANG II levels (150
fmol/mg protein), as well as secreted ANG II in the media (30
fmol/mg protein after 24 h) using a Peninsula ELISA. Basal
ANG II levels were also similar (180 fmol/mg protein) in
immortalized mouse podocytes, and peptide content increased
with mechanical strain (38). Singh and colleagues (132) quantified ANG II content at 200 fmol/mg protein in cultured
neonatal cardiomyocytes and fibroblasts; the cellular ANG II
levels were also markedly elevated by high glucose conditions
and exhibited a greater nuclear localization for ANG II. ANG
II content was determined by a direct Peninsula ELISA, and
the relative peptide expression in control and high glucoseexposed cardiomyocytes was confirmed by antibody capture
and HPLC-MS (132). Finally, we assessed the effect of advanced glycation end products on angiotensin expression in
renal NRK-52E cells (9). Basal cellular content was quantified

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ASSESSMENT OF THE RAS

by direct RIAs for ANG I (85 fmol/mg protein; Peninsula),


ANG II (150 fmol/mg protein; ALPCO), and ANG-(17) (400
fmol/mg protein; custom RIA) (9). Advanced glycation end
product exposure significantly reduced ANG-(17) and increased metabolism of the peptide that was associated with
myofibroblast transition but did not impact ANG II or ANG I
levels (9). As these cells express an intracellular RAS, nuclear
content of ANG II and ANG-(17) was quantified at 60
fmol/mg protein consistent with nuclear immunostaining for
both peptides (10).
In review of the cell studies, angiotensin content in various
cell types is 200 fmol/mg protein (Table 1). The high
angiotensin levels compared with tissues likely reflect the
lower cellular protein to normalize peptide content, as well as
the greater density of angiotensin-expressing cells maintained
in culture; however, direct comparisons of angiotensins quantified by RIA (or ELISA) to HPLC-RIA and LC-MS are
lacking, and the true cellular content of ANG II or ANG-(17)
in these cell models is not known. A second issue with cell
studies is the presence of circulating RAS components in fetal
bovine serum (10). Cells can be maintained in low or serumfree conditions for short periods, but internalized angiotensinogen or renin may persist and contribute to the intracellular
expression of angiotensin peptides, particularly if nonrenin
proteases that can process the precursor (cathepsins, tonin) are
also expressed. Furthermore, bovine angiotensinogen contains
an Ile5 substitution for Val5 in the NH2-terminal portion of the
protein, and RIAs will not distinguish these angiotensin isoforms unless samples are initially fractionated by HPLC or
characterized by LC-MS (130). ANG II also undergoes internalization through both AT1R-dependent and -independent
pathways that may further contribute to the cellular content of
the peptide (40, 92, 140, 150). Indeed, the interaction between
the circulating RAS components and the cellular or tissue
system is likely a dynamic process in vivo that should be
examined to a greater extent in cell studies. Finally, the
subcellular compartmentalization of angiotensin peptides and
other components is becoming increasingly evident (1, 2, 25,
35, 40, 54, 74, 140, 151). Apart from establishing the functional role of these intracellular systems, a greater emphasis
should be placed on the quantification of angiotensins in
cellular compartments, and particularly the extent that altered
peptide content in tissue reflects discrete changes in the subcellular systems such as the nucleus and mitochondria.
Conclusions
The current review assessed the biochemical approaches to
quantify the multiple components of the RAS, as well as
attempt a consensus on expected values of endogenous angiotensin peptides in the circulation, tissues, and cells. In regard to
the peptidase and receptor components of the RAS, an optimal
approach should reflect some measure of the activity of these
proteins, although it is acknowledged that the receptor expression is an order of magnitude lower than that of other RAS
proteins and presents a more difficult challenge for receptor
characterization This same issue holds for true for the quantification of angiotensin peptides given their very low abundance; optimal approaches should reflect a highly sensitive and
specific method coupled with relatively high throughput to
evaluate large sample sets. The LC-MS/MS approach appears

to be the ideal choice to quantify angiotensin peptides; however, the required expertise and overall expense of these
systems limits their widespread availability to a limited number
of laboratories at the current time. Moreover, the application of
MS to the determination of angiotensins does not necessarily
guarantee an accurate profile or an absolute value in plasma
and tissues. Thus RIA or ELISA methods will continue to be
the predominant approach to quantify angiotensin peptides and
assess alterations in peptide expression. The limitations of
these assays, as well as the methods applied for sampling
handling, extraction, and approaches for validation, must be
considered for the accurate assessment of the peptide hormones
of the RAS, as well as to establish that an altered phenotypic or
treatment response truly reflects changes in peptide expression.
Importantly, validation of these assays for either individual
samples or a sample pool by HPLC-RIA should be a requirement for the analysis of endogenous angiotensin peptides in
various compartments, and particularly in studies that assess
the cellular expression of angiotensins.
ACKNOWLEDGMENTS
I gratefully acknowledge K. Bridget Brosnihan and Debra I. Diz of the
Hypertension and Vascular Research Center for their critical reading and
insightful comments pertaining to this review.
GRANTS
This study was supported in part by National Institute of Health Grants
HL-56973, HL-51952, HD-084227, HD-047584, and HD-017644 and American Heart Association Grants AHA-151521 and AHA-355741. An unrestricted grant from the Farley-Hudson Foundation (Jacksonville, NC), Groskert
Heart Fund, and the Wake Forest Venture Fund is also acknowledged.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
M.C.C. drafted, edited, revised, and approved final version of manuscript.
REFERENCES
1. Abadir PM, Foster DB, Crow M, Cooke CA, Rucker JJ, Jain A,
Smith BJ, Burks TN, Cohn RD, Fedarko NS, Carey RM, ORourke
B, Walston JD. Identification and characterization of a functional mitochondrial angiotensin system. Proc Natl Acad Sci USA 108: 14849
14854, 2011.
2. Abadir PM, Walston JD, Carey RM. Subcellular characteristics of
functional intracellular renin-angiotensin systems. Peptides 38: 437445,
2012.
3. Ahmad S, Simmons T, Varagic J, Moniwa N, Chappell MC, Ferrario
CM. Chymase-dependent generation of angiotensin II from angiotensin(112) in human atrial tissue. PLoS One 6: e28501, 2011.
4. Albiston AL, Fernando RN, Yeatman HR, Burns P, Ng L, Daswani
D, Diwakarla S, Pham V, Chai SY. Gene knockout of insulin-regulated
aminopeptidase: loss of the specific binding site for angiotensin IV and
age-related deficit in spatial memory. Neurobiol Learn Mem 93: 19 30,
2010.
5. Alge JL, Karakala N, Neely BA, Janech MG, Tumlin JA, Chawla LS,
Shaw AD, Arthur JM. Association of elevated urinary concentration of
renin-angiotensin system components and severe AKI. Clin J Am Soc
Nephrol 8: 20432052, 2013.
6. Alghamri MS, Morris M, Meszaros JG, Elased KM, Grobe N. Novel
role of aminopeptidase-A in angiotensin-(17) metabolism post myocardial infarction. Am J Physiol Heart Circ Physiol 306: H1032H1040,
2014.
7. Ali Q, Wu Y, Nag S, Hussain T. Estimation of angiotensin peptides in
biological samples by LC/MS method. Anal Methods 6: 215222, 2014.

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org

Review
ASSESSMENT OF THE RAS
8. Ali Q, Wu Y, Hussain T. Chronic AT2 receptor activation increases
renal ACE2 activity, attenuates AT1 receptor function and blood pressure
in obese Zucker rats. Kidney Int 84: 931939, 2013.
9. Alzayadneh EM, Chappell MC. Angiotensin-(17) abolishes AGEinduced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2. Cell Signal 26: 30273035, 2014.
10. Alzayadneh EM, Chappell MC. Nuclear expression of renin-angiotensin system components in NRK-52E renal epithelial cells. J Renin
Angiotensin Aldosterone Syst. 2014 Jun 24. pii: 1470320313515039.
[Epub ahead of print].
11. Anderson NL, Anderson NG. The human proteonome. Mol Cell Proteonomics 1: 845867, 2002.
12. Bader M, Alenina N, Andrade-Navarro MA, Santos RA. MAS and its
related G protein-coupled receptors, Mrgprs. Pharmacol Rev 66: 1080
1105, 2014.
13. Baker M. Reproducibility crisis: blame it on the antibodies. Nature 521:
274 276, 2015.
14. Balcells E, Meng QC, Hageman GR, Palmer RW, Durand JN,
DellItalia LJ. Angiotensin II formation in dog heart is mediated by
different pathways in vivo and in vitro. Am J Physiol 40: H417H421,
1996.
15. Bernstein KE, Ong FS, Blackwell WL, Shah KH, Giani JF, Gonzalez-Villalobos RA, Shen XZ, Fuchs S, Touyz RM. A modern understanding of the traditional and nontraditional biological functions of
angiotensin-converting enzyme. Pharmacol Rev 65: 146, 2013.
16. Bertoncello N, Moreria RP, Arita DY, Aragao DS, Watanabe IKM,
Dantas PS, Santos R, Mattar-Rosa R, Yokota R, Cunha TS, Casarini
DE. Diabetic nephropathy induced by Ace gene dosage is associated with
high renal levels of angiotensin-(17) and bradykinin. J Diabetes Res 67:
113, 2015.
17. Bkaily G, Nader M, Avedanian L, Choufani S, Jacques D,
DOrleans-Juste P, Gobeil F, Chemtob S, Al-Khoury J. G-proteincoupled receptors, channels, and Na-H exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cells.
Can J Physiol Pharmacol 84: 431441, 2006.
18. Bodineau L, Frugiere A, Marc Y, Inguimbert N, Fassot C, Balavoine
F, Roques B, Llorens-Cortes C. Orally active aminopeptidase A inhibitors reduce blood pressure: a new strategy for treating hypertension.
Hypertension 51: 1318 1325, 2008.
19. Burger D, Reudelhuber TL, Mahajan A, Chibale K, Sturrock ED,
Touyz RM. Effects of a domain-selective ACE inhibitor in a mouse
model of chronic angiotensin II-dependent hypertension. Clin Sci (Lond)
127: 5763, 2014.
20. Camenzind AG, van der Gugten JG, Popp R, Holmes DT, Borchers
CH. Development and evaluation of an immuno-MALDI (iMALDI)
assay for angiotensin I and the diagnosis of secondary hypertension. Clin
Proteomics 10: 20, 2013.
21. Campbell DJ. Angiotensin II generation in vivo: does it involve enzymes other than renin and angiotensin-converting enzyme? J Renin
Angiotensin Aldosterone Syst 13: 314 316, 2012.
22. Campbell DJ, Duncan AM, Kladis A, Harrap SB. Angiotensin peptides in spontaneously hypertensive and normotensive donryu rats. Hypertension 25: 928 934, 1995.
23. Campbell DJ, Kladis A, Duncan AM. Nephrectomy, converting enzyme inhibition and angiotensin peptides. Hypertension 22: 513522,
1993.
24. Campbell DJ, Alexiou T, Xiao HD, Fuchs S, McKinley MJ, Corvol P,
Bernstein KE. Effect of reduced ACE gene expression and ACE inhibition on angiotensin and bradykinin peptide levels in mice. Hypertension 43: 854 859, 2004.
25. Carey RM. Functional intracellular renin-angiotensin systems: potential
for pathophysiology of disease. Am J Physiol Regul Integr Comp Physiol
302: R479 R481, 2012.
26. Castro-Moreno P, Pardo JP, Henandez-Munoz R, Lopez-Guerrero
JJ, Valle-Mondragon LD, Pastelin-Hernandez G, Ibarra-Barajas M,
Vilalobos-Molina R. Captopril avoids hypertension, the increase in
plasma angiotensin II but increases angiotensin 17 and angiotensin
II-induced perfusion pressure in isolated kidney in SHR. Auton Autacoid
Pharmacol 32: 6169, 2012.
27. Caughey GH, Raymond WW, Wolters PJ. Angiotensin II generation
by mast cell alpha- and beta-chymases. Biochim Biophys Acta 1480:
245257, 2000.
28. Caughey GH. Mast cell proteases as protective and inflammatory mediators. Adv Exp Med Biol 716: 212234, 2011.

H149

29. Chappell MC. Emerging evidence for a functional angiotensin-converting enzyme 2-angiotensin-(17) mas receptor axis; more than regulation
of blood pressure? Hypertension 50: 596 599, 2007.
30. Chappell MC, Brosnihan KB, Diz DI, Ferrario CM. Identification of
angiotensin-(17) in rat brain: evidence for differential processing of
angiotensin peptides. J Biol Chem 264: 16518 16523, 1989.
31. Chappell MC, Gomez MN, Pirro NT, Ferrario CM. Release of
angiotensin-(17) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition. Hypertension 35: 348 352, 2000.
32. Chappell MC, Tallant EA, Brosnihan KB, Ferrario CM. Conversion
of angiotensin I to angiotensin-(17) by thimet oligopeptidase (EC
3.42415) in vascular smooth muscle cells. J Vasc Med Biol 5: 129 137,
1994.
33. Coelho MS, Lpes KL, de Aquino Frietas R, de Oliveira-Sales EB,
Bergasmaschi CT, Campos RB, Casarinin DE, Camona AK, da Silva
Arauo M., Heimann JC, Dolnikoff MS. High sucrose intake in rats is
associated with increased ACE2 and angiotensin-(17) levels in adipose
tissue. Regul Pept 162: 6167, 2010.
34. Cohen JA, Lindsey SH, Pirro NT, Brosnihan KB, Gallagher PE,
Chappell MC. Influence of estrogen depletion and salt loading on renal
angiotensinogen expression in the mRen(2). Lewis strain. Am J Physiol
Renal Physiol 299: F35F42, 2010.
35. Cook JL, Re RN. Lessons from in vitro studies and a related intracellular
angiotensin II transgenic mouse model. Am J Physiol Regul Integr Comp
Physiol 302: R482R493, 2012.
36. DellItalia LJ, Meng QC, Balcells E, Wei CC, Palmer R, Hageman
GR, Durand J, Hankes GH, Oparil S. Compartmentalization of angiotensin II generation in the dog heart. Evidence for independent mechanisms in intravascular and interstitial spaces. J Clin Invest 100: 253258,
1997.
37. DeSilva PE, Husain A, Smeby RR, Khairallah PA. Measurements of
immunoreactive angiotensin peptides in rat tissues: some pitfalls in
angiotensin II analysis. Anal Biochem 174: 80 87, 1988.
38. Durvasula RV, Petermann AT, Hiromura K, Blonski M, Pippin J,
Mundel P, Pichler R, Griffin S, Couser WG, Shankland SJ. Activation of a local tissue angiotensin system in podocytes by mechanical
strain. Kidney Int 65: 30 39, 2004.
39. Elased KM, Cool DR, Morris M. Novel mass spectrometric methods
for evaluation of plasma angiotensin converting enzyme 1 and renin
activity. Hypertension 46: 953959, 2005.
40. Ellis B, Li XC, Miguel-Qin E, Gu V, Zhuo JL. Evidence for a
functional intracellular angiotensin system in the proximal tubule of the
kidney. Am J Physiol Regul Integr Comp Physiol 302: R494 R509,
2012.
41. Epelman S, Shrestha K, Troughton RW, Francis GS, Sen S, Klein
AL, Tang WH. Soluble angiotensin-converting enzyme 2 in human heart
failure: relation with myocardial function and clinical outcomes. J Card
Fail 15: 565571, 2009.
42. Fernandes T, Hashimoto NY, Magalhaes FC, Fernandes FB, Casarini DE, Carmona AK, Krieger JE, Phillips MI, Oliveira EM.
Aerobic exercise training-induced left ventricular hypertrophy involves
regulatory MicroRNAs, decreased angiotensin-converting enzyme-angiotensin ii, and synergistic regulation of angiotensin-converting enzyme
2-angiotensin (17). Hypertension 58: 182189, 2011.
43. Ferrario CM. Angiotensin-converting enzyme 2 and angiotensin-(17):
an evolving story in cardiovascular regulation. Hypertension 45: 313
320, 2006.
44. Ferrario CM, Jessup JA, Gallagher PE, Averill DB, Brosnihan KB,
Tallant EA, Smith RD, Chappell MC. Effects of renin angiotensin
system blockade on renal angiotensin-(17) forming enzymes and receptors. Kidney Int 68: 2189 2196, 2005.
45. Ferrario CM, Martell N, Yunis C, Flack JM, Chappell MC, Brosnihan KB, Dean RH, Fernandez A, Novikov SV, Pinillas C, Luque M.
Characterization of angiotensin-(17) in the urine of normal and essential
hypertensive subjects. Am J Hypertens 11: 137146, 1998.
46. Ferrario CM, Strawn WB. Role of the renin-angiotensin-aldosterone
system and proinflammatory mediators in cardiovascular disease. Am J
Cardiol 98: 121128, 2006.
47. Ferreira AJ, Mura TM, Fraga-Silva RA, Castro CH, Raizada MK,
Santos RA. New cardiovascular and pulmonary therapeutic strategies
based on the Angiotensin-converting enzyme 2/angiotensin-(17)/mas
receptor axis. Int J Hypertens 2012: 147825, 2012.
48. Franco MC, Akamine EH, Di Marco GS, Casarini DE, Fortes ZB,
Tostes RC, Carvalho MH, Nigro D. NADPH oxidase and enhanced

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org

Review
H150

49.

50.

51.

52.
53.

54.

55.

56.

57.

58.

59.

60.

61.

62.
63.

64.

65.

66.

67.

ASSESSMENT OF THE RAS

superoxide generation in intrauterine undernourished rats: involvement


of the renin-angiotensin system. Cardiovasc Res 59: 767775, 2003.
Garabelli PJ, Modrall JG, Penninger JM, Ferrario CM, Chappell
MC. Distinct roles for angiotensin converting enzyme 2 and carboxypeptidase A in the processing of angiotensins in the murine heart. Exp
Physiol 95: 613621, 2008.
Gilliam-Davis S, Payne VS, Kasper SO, Tommasi EN, Robbins ME,
Diz DI. Long-term AT1 receptor blockade improves metabolic function
and provides renoprotection in Fischer-344 rats. Am J Physiol Heart Circ
Physiol 293: H1327H1333, 2007.
Gomes-Santos IL, Fernandes T, Couto GK, Ferreira-Filho JC, Salemi VM, Fernandes FB, Casarini DE, Brum PC, Rossoni LV, de
Oliveira EM, Negrao CE. Effects of exercise training on circulating and
skeletal muscle renin-angiotensin system in chronic heart failure rats.
PLoS One 9: e98012, 2014.
Gonzalez AA, Prieto MC. Roles of collecting duct renin and prorenin
receptor in hypertension. Ther Adv Cardiovasc Dis 9: 191200, 2015.
Grobe N, Elased KM, Cool DR, Morris M. Mass spectrometry for the
molecular imaging of angiotensin metabolism in kidney. Am J Physiol
Endocrinol Metab 302: E1016 E1024, 2012.
Gwathmey TM, Alzayadneh EM, Pendergrass KD, Chappell MC.
Novel roles of nuclear angiotensin receptors and signaling mechanisms.
Am J Physiol Regul Integr Comp Physiol 302: R518 R530, 2012.
Gwathmey TM, Pendergrass KD, Reid SD, Rose JC, Diz DI, Chappell MC. Angiotensin-(17)-angiotensin-converting enzyme 2 attenuates
reactive oxygen species formation to angiotensin II within the cell
nucleus. Hypertension 55: 166 171, 2010.
Gwathmey TM, Shaltout HA, Pendergrass KD, Pirro NT, Figueroa
JP, Rose JC, Diz DI, Chappell MC. Nuclear angiotensin II type 2 (AT2)
receptors are functionally linked to nitric oxide production. Am J Physiol
Renal Physiol 296: F1484 F1493, 2009.
Gwathmey TM, Shaltout HA, Rose JC, Diz DI, Chappell MC.
Glucocorticoid-induced fetal programming alters the functional complement of angiotensin receptor subtypes within the kidney. Hypertension
57: 620 626, 2011.
Gwathmey TM, Westwood BM, Pirro NT, Tang L, Rose JC, Diz DI,
Chappell MC. Nuclear angiotensin-(17) receptor is functionally coupled to the formation of nitric oxide. Am J Physiol Renal Physiol 299:
F983F990, 2010.
Hafko R, Villapol S, Nostramo R, Symes A, Sabban EL, Inagami T,
Saavedra JM. Commercially available angiotensin II At(2) receptor
antibodies are nonspecific. PLoS One 8: e69234, 2013.
Haschke M, Schuster M, Poglitsch M, Loibner H, Salzberg M,
Bruggisser M, Penninger J, Krahenbuhl S. Pharmacokinetics and
pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects. Clin Pharmacokinet 52: 783792,
2013.
Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard
LM, Coffman TM. Lack of specificity of commercial antibodies leads to
misidentification of angiotensin type 1 receptor protein. Hypertension 61:
253258, 2013.
Hildebrand D, Merkel P, Eggers LF, Schluter H. Proteolytic processing of angiotensin-I in human blood plasma. PLoS One 8: e64027, 2013.
Hilgers KF, Bingener E, Stumpf C, Muller DM, Schmieder RE,
Veelken R. Angiotensinases restrict locally generated angiotensin II to
the blood vessel wall. Hypertension 31: 368 372, 1998.
Inoue H, Fukui K, Takashi S, Miyake Y. Genetic and molecular
properties of the human and rat renin binding protein with reference to
the function of the leucine zipper motif. J Biochem 110: 493500, 1991.
Ishigami T, Kino T, Chen L, Minegishi S, Araki N, Umemura M, Abe
K, Sasaki R, Yamana H, Umemura S. Identification of bona fide
alternative renin transcripts expressed along cortical tubules and potential
roles in promoting insulin resistance in vivo without significant plasma
renin activity elevation. Hypertension 64: 125133, 2014.
Jankowski V, Tolle M, Santos RA, Gunthner T, Krause E, Beyermann M, Welker P, Bader M, Pinheiro SV, Sampaio WO, Lautner
R, Kretschmer A, van der Giet M, Zidek W, Jankowski J. Angioprotectin: an angiotensin II-like peptide causing vasodilatory effects.
FASEB J 25: 29872995, 2011.
Jankowski V, Vanholder R, van der Giet M, Tolle M, Karadogan S,
Gobom J, Furkert J, Oksche A, Krause E, Tran TN, Tepel M,
Schuchardt M, Schluter H, Wiedon A, Beyermann M, Bader M,
Todiras M, Zidek W, Jankowski J. Mass-spectrometric identification

68.

69.

70.

71.
72.

73.

74.
75.

76.
77.
78.

79.
80.
81.
82.

83.

84.
85.

86.

87.

of a novel angiotensin peptide in human plasma. Arterioscler Thromb


Vasc Biol 27: 297302, 2007.
Kemp BA, Bell JF, Rottkamp DM, Howell NL, Shao W, Navar LG,
Padia SH, Carey RM. Intrarenal angiotensin III is the predominant
agonist for proximal tubule angiotensin type 2 receptors. Hypertension
60: 387395, 2012.
Kobori H, Alper AB Jr, Shenava R, Katsurada A, Saito T, Ohashi N,
Urushihara M, Miyata K, Satou R, Hamm LL and Navar LG.
Urinary angiotensinogen as a novel biomarker of the intrarenal reninangiotensin system status in hypertensive patients. Hypertension 53:
344 350, 2009.
Kobori H, Katsurada A, Miyata K, Ohashi N, Satou R, Saito T,
Hagiwara Y, Miyashita K, Navar LG. Determination of plasma and
urinary angiotensinogen levels in rodents by newly developed ELISA.
Am J Physiol Renal Physiol 294: F1257F1263, 2008.
Kobori H, Nangaku M, Navar LG, Nishiyama A. The intrarenal
renin-angiotensin system: from physiology to the pathobiology of hypertension and kidney disease. Pharmacol Rev 59: 251287, 2007.
Kobori H, Ohashi N, Katsurada A, Miyata K, Satou R, Saito T,
Yamamoto T. Urinary angiotensinogen as a potential biomarker of
severity of chronic kidney diseases. J Am Soc Hypertens 2: 349 354,
2008.
Kohara K, Tabuchi Y, Senanayake P, Brosnihan KB, Ferrario CM.
Reassessment of plasma angiotensins measurement: effects of protease
inhibitors and sample handling procedures. Peptides 12: 11351141,
1991.
Kumar R, Thomas CM, Yong QC, Chen W, Baker KM. The intracrine renin-angiotensin system. Clin Sci (Lond) 123: 273284, 2012.
Lautner RQ, Villela DC, Fraga-Silva RA, Silva N, Verano-Braga T,
Costa-Fraga F, Jankowski J, Jankowski V, Sousa F, Alzamora A,
Soares E, Barbosa C, Kjeldsen F, Oliveira A, Braga J, Savergnini S,
Maia G, Peluso AB, Passos-Silva D, Ferreira A, Alves F, Martins A,
Raizada M, Paula R, Motta-Santos D, Klempin F, Pimenta A,
Alenina N, Sinisterra R, Bader M, Campagnole-Santos MJ, Santos
RA. Discovery and characterization of alamandine: a novel component
of the renin-angiotensin system. Circ Res 112: 1104 1111, 2013.
Lavoie JL, Liu X, Bianco RA, Beltz TG, Johnson AK, Sigmund CD.
Evidence supporting a functional role for intracellular renin in the brain.
Hypertension 47: 461466, 2006.
Lavrentyev EN, Estes AM, Malik KU. Mechanism of high glucose
induced angiotensin II production in rat vascular smooth muscle cells.
Circ Res 101: 455464, 2007.
Lawrence AC, Evin G, Kladis A, Campbell DJ. An alternative strategy
for the radioimmunoassay of angiotensin peptides using amino-terminaldirected antisera: measurement of eight angiotensin peptides in human
plasma. J Hypertens 8: 715724, 1990.
Lew RA. HPLC in the analysis of peptide metabolism. Methods Mol Biol
251: 275290, 2004.
Lew RA, Mustafa T, Ye S, McDowall SG, Chai SY, Albiston AL.
Angiotensin AT4 ligands are potent, competitive inhibitors of insulin
regulated aminopeptidase (IRAP). J Neurochem 86: 344 350, 2003.
Lew RA, Tochon-Danguy N, Hamilton CA, Stewart KM, Aguilar
MI, Smith AI. Quenched fluorescent substrate-based peptidase assays.
Methods Mol Biol 298: 143150, 2005.
Liu L, Gonzalez AA, McCormack M, Seth DM, Kobori H, Navar
LG, Prieto MC. Increased renin excretion is associated with augmented
urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats. Am J Physiol Renal Physiol 301: F1195F1201, 2011.
Lortie M, Bark S, Blantz R, Hook V. Detecting low-abundance
vasoactive peptides in plasma: progress toward absolute quantitation
using nano liquid chromatography-mass spectrometry. Anal Biochem
394: 164 170, 2009.
Lumbers ER, Skinner SL. Origin of renin activity in human urine. Circ
Res 24:689 697 1969.
Mazzolai L, Pedrazzini T, Nicoud F, Gabbiani G, Brunner HR,
Nussberger J. Increased cardiac angiotensin II levels induce right and
left ventricular hypertrophy in normotensive mice. Hypertension 35:
985991, 2000.
Miners JS, Verbeek MM, Rikkert MO, Kehoe PG, Love S. Immunocapture-based fluorometric assay for the measurement of neprilysinspecific enzyme activity in brain tissue homogenates and cerebrospinal
fluid. J Neurosci Methods 167: 229 236, 2008.
Mizuno K, Tani M, Niimura S, Hashimoto S, Satoh A, Shimamoto K,
Inagami T, Fukuchi S. Direct evidence for local generation and release

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org

Review
ASSESSMENT OF THE RAS

88.

89.

90.

91.
92.

93.

94.
95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

of angiotensin II in human vascular tissue. Biochem Biophys Res Commun 165: 457463, 1989.
Modrall JG, Sadjadi J, Brosnihan KB, Gallagher PE, Ya CH,
Kramer GL, Bernstein KE, Chappell MC. Depletion of tissue ace
differentially influences the intrarenal and urinary expression of angiotensins. Hypertension 43: 4849 4853, 2003.
Nagata S, Hatakeyama K, Asami M, Tokashiki M, Hibino H, Nishiuchi Y, Kuwasako K, Kato J, Asada Y, Kitamura K. Big angiotensin25: a novel glycosylated angiotensin-related peptide isolated from human
urine. Biochem Biophys Res Commun 441: 757762, 2013.
Nagata S, Kato J, Sasaki K, Minamino N, Eto T, Kitamura K.
Isolation and identification of proangiotensin-12, a possible component
of the renin-angiotensin system. Biochem Biophys Res Commun 350:
1026 1031, 2006.
Navar LG, Imig JD, Zou L, Wang CT. Intrarenal production of
angiotensin II. Semin Nephrol 17: 412422, 1997.
Navar LG, Kobori H, Prieto MC, Gonzalez-Villalobos RA. Intratubular renin-angiotensin system in hypertension. Hypertension 57: 355
362, 2011.
Navar LG, Prieto MC, Satou R, Kobori H. Intrarenal angiotensin II
and its contribution to the genesis of chronic hypertension. Curr Opin
Pharmacol 11: 180 186, 2011.
Nguyen G. Renin/prorenin receptors. Kidney Int 69: 15031506, 2006.
Nishiyama A, Seth DM, Navar LG. Renal interstitial fluid angiotensin
I and angiotensin II concentrations during local angiotensin-converting
enzyme inhibition. J Am Soc Nephrol 13: 22072212, 2002.
Nishiyama A, Seth DM, Navar LG. Renal interstitial fluid concentrations of angiotensins I and II in anesthetized rats. Hypertension 39:
129 134, 2002.
Nussberger J, Brunner DB, Nyfeler JA, Linder L, Brunner HR.
Measurement of immunoreactive angiotensin-(17) heptapeptide in human blood. Clin Chem 47: 726 729, 2001.
Nussberger J, Brunner DB, Waeber B, Brunner HR. True versus
immunoreactive angiotensin II in human plasma. Hypertension 7, Suppl
I: I-1I-7, 1985.
Nussberger J, Buhler K, Waeber B, Brunner HR. Identification and
quantitation of angiotensins. J Cardiovasc Pharmacol 8, Suppl 10:
S23S28, 1986.
Nussberger J, Wuerzner G, Jensen C, Brunner HR. Angiotensin II
suppression in humans by the orally active renin inhibitor Aliskiren
(SPP100): comparison with enalapril. Hypertension 39: E1E8, 2002.
Ocaranza MP, Michea L, Chiong M, Lagos CF, Lavandero S, Jalil
JE. Recent insights and therapeutic perspectives of angiotensin-(19) in
the cardiovascular system. Clin Sci (Lond) 127: 549 557, 2014.
Olkowicz M, Radulska A, Suraj J, Kij A, Walczak M, Chlopicki S,
Smolenski RT. Development of a sensitive, accurate and robust liquid
chromatography/mass spectrometric method for profiling of angiotensin
peptides in plasma and its application for atherosclerotic mice. J Chromatogr A 1393: 3746, 2015.
Padia SH, Kemp BA, Howell NL, Gildea JJ, Keller SR, Carey RM.
Intrarenal angiotensin III infusion induces natriuresis and angiotensin
type 2 receptor translocation in Wistar-Kyoto but not in spontaneously
hypertensive rats. Hypertension 53: 338 343, 2009.
Pendergrass KD, Averill DB, Ferrario CM, Diz DI, Chappell MC.
Differential expression of nuclear AT1 receptors and angiotensin II
within the kidney of the male congenic mRen2. Lewis rat. Am J Physiol
Renal Physiol 290: F1497F1506, 2006.
Pendergrass KD, Gwathmey TM, Michalek RD, Grayson JM, Chappell MC. The angiotensin II-AT1 receptor stimulates reactive oxygen
species within the cell nucleus. Biochem Biophys Res Commun 384:
149 154, 2009.
Peters J, Clausmeyer S. Intracellular sorting of renin: cell type specific
differences and their consequences. J Mol Cell Cardiol 34: 15611568,
2002.
Peters J, Wanka H, Peters B, Hoffmann S. A renin transcript lacking
exon 1 encodes for a non-secretory intracellular renin that increases
aldosterone production in transgenic rats. J Cell Mol Med 12: 1229
1237, 2008.
Poglitsch M, Domenig O, Schwager C, Stranner S, Peball B, Janzek
E, Wagner B, Jungwirth H, Loibner H, Schuster M. Recombinant
expression and characterization of human and murine ACE2: Speciesspecific activation of the alternative renin-angiotensin-System. Int J
Hypertens 2012: 428950, 2012.

H151

109. Reaux-Le GA, Iturrioz X, Fassot C, Claperon C, Roques BP, Llorens-Cortes C. Role of angiotensin III in hypertension. Curr Hypertens
Rep 7: 128 134, 2005.
110. Ribeiro AA, Palomino Z, Lima MP, Souza LE, Ferreira DS, Pesquero JB, Irigoyen MC, Pesquero JL, Casarini DE. Characterization
of the renal renin-angiotensin system in transgenic mice that express rat
tonin. J Renin Angiotensin Aldosterone Syst. 2015 Jul 27. pii:
1470320315595572. [Epub ahead of print].
111. Rice GI, Jones AL, Grant PJ, Carter AM, Turner AJ, Hooper NM.
Circulating activities of angiotensin-converting enzyme, its homolog,
angiotensin-converting enzyme 2, and neprilysin in a family study.
Hypertension 48: 914 920, 2006.
112. Rice GI, Thomas DA, Grant PJ, Turner AJ, Hooper NM. Evaluation
of angiotensin-converting enzyme (ACE), its homologue ACE2 and
neprilysin in angiotensin peptide metabolism. Biochem J 383: 4551,
2004.
113. Roksnoer LC, Verdonk K, van den Meiracker AH, Hoorn EJ, Zietse
R, Danser AH. Urinary markers of intrarenal renin-angiotensin system
activity in vivo. Curr Hypertens Rep 15: 8188, 2013.
114. Ronchi FA, Irigoyen MC, Casarini DE. Association of somatic and
N-domain angiotensin-converting enzymes from Wistar rat tissue with
renal dysfunction in diabetes mellitus. J Renin Angiotensin Aldosterone
Syst 8: 34 41, 2007.
115. Rubio-Ruiz ME, Del Valle-Mondragon L, Castregjon-Tellez V,
Carreon-Torees E, Diaz-Diaz E, Guarner-Lans V. Angiotensin II and
17 during aging in metabolic syndrome rats Expression of AT1 and AT2
and Mas receptors in abdominal white adipose tissue. Peptides 57:
101108, 2014.
116. Rykl J, Thiemann J, Kurzawski S, Pohl T, Gobom J, Zidek W,
Schluter H. Renal cathepsin G and angiotensin II generation. J Hypertens 24: 17971807, 2006.
117. Santos CF, Caprio MA, Oliveira EB, Salgado MC, Schippers DN,
Munzenmaier DH, Greene AS. Functional role, cellular source, and
tissue distribution of rat elastase-2, an angiotensin II-forming enzyme.
Am J Physiol Heart Circ Physiol 285: H775H783, 2003.
118. Santos CF, Greene AS, Salgado MC, Oliveira EB. Conversion of renin
substrate tetradecapeptide to angiotensin II by rat MAB elastase-2. Can
J Physiol Pharmacol 82: 1000 1005, 2004.
119. Santos RA. Angiotensin-(17). Hypertension 63: 1138 114, 2014.
120. Santos RA, Ferreira AJ, Verano-Braga T, Bader M. Angiotensinconverting enzyme 2, angiotensin-(17) and Mas: new players of the
renin-angiotensin system. J Endocrinol 216: R1R17, 2013.
121. Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de
Bul I, Heringer-Walther S, Pinheiro SV, Lopes MT, Bader M,
Mendes EP, Lemos VS, Campagnole-Santos MJ, Schultheiss H-P,
Speth R, Walther T. Angiotensin-(17) is an endogenous ligand for the
G protein-coupled receptor Mas. Proc Natl Acad Sci USA 100: 8258
8263, 2003.
122. Santos RAS, Krieger EM, Greene LJ. An improved fluorometric assay
of rat serum and plasma converting enzyme. Hypertension 7: 244 252,
1985.
123. Schechter NM, Irani AA, Sprows JL, Abernethy J, Winstroub BU,
Schwartz LB. Identification of a cathepsin G-like proteinase in the
MCTC type of human mast cell. J Immunol 145: 26522661, 1990.
124. Schuijt MP, van Kats JP, de ZS, Duncker DJ, Verdouw PD,
Schalekamp MA, Danser AH. Cardiac interstitial fluid levels of angiotensin I and II in the pig. J Hypertens 17: 18851891, 1999.
125. Schulz A, Jankowski J, Zidek W, Jankowski V. Absolute quantification of endogenous angiotensin II levels in human plasma using ESILC-MS/MS. Clin Proteomics 11: 37, 2014.
126. Semaan W, Desbiens L, Houde M, Labonte J, Gagnon H, Yamamoto
D, Takai S, Laidlaw T, Bkaily G, Schwertani A, Pejler G, Levesque
C, Desjardins R, Day R, DOrleans-Juste P. Chymase inhibitorsensitive synthesis of endothelin-1 (131) by recombinant mouse mast
cell protease 4 and human chymase. Biochem Pharmacol 94: 91100,
2015.
127. Senanayake PD, Moriguchi A, Kumagai H, Ganten D, Ferrario CM,
Brosnihan KB. Increased expression of angiotensin peptides in the brain
of transgenic hypertensive rats. Peptides 15: 919 926, 1994.
128. Senanayake PS, Smeby RR, Martins AS, Moriguchi A, Kumagai H,
Ganten D, Brosnihan KB. Adrenal, kidney, and heart angiotensins in
female murine Ren-2 transfected hypertensive rats. Peptides 19: 1685
1694, 1998.

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org

Review
H152

ASSESSMENT OF THE RAS

129. Shaltout HA, Westwood B, Averill DB, Ferrario CM, Figueroa J, Diz
DI, Rose JC, Chappell MC. Angiotensin metabolism in renal proximal
tubules, urine and serum of sheep: Evidence for ACE2-dependent processing of Angiotensin II. Am J Physiol Renal Physiol 292: F82F91,
2007.
130. Shao W, Seth DM, Navar LG. Augmentation of endogenous intrarenal
angiotensin II levels in Val5-Ang II-infused rats. Am J Physiol Renal
Physiol 296: F1067F1071, 2009.
131. Shi Y, Lo CS, Padda R, Abdo S, Chenier I, Filep JG, Ingelfinger JR,
Zhang SL, Chan JS. Angiotensin-(17) prevents systemic hypertension,
attenuates oxidative stress and tubulointerstitial fibrosis, and normalizes
renal angiotensin-converting enzyme 2 and Mas receptor expression in
diabetic mice. Clin Sci (Lond) 128: 649 663, 2015.
132. Singh VP, Le B, Bhat VB, Baker KM, Kumar R. High-glucoseinduced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes. Am J Physiol Heart Circ Physiol 293:
H939 H948, 2007.
133. Suski M, Gebska A, Olszanecki R, Stachowicz A, Uracz D, Madej J,
Korbut R. Influence of atorvastatin on angiotensin I metabolism in
resting and TNF-alpha -activated rat vascular smooth muscle cells. J
Renin Angiotensin Aldosterone Syst 15: 378 383, 2014.
134. Te RL, van Esch JH, Roks AJ, van den Meiracker AH, Danser AH.
Hypertension: renin-angiotensin-aldosterone system alterations. Circ Res
116: 960 975, 2015.
135. Tenorio-Lopez FA, Zarco-Olvera G, Sanchez-Mendoza A, RosasPeralta M, Pastelin-Hernandez G, Del Valle-Mondragon L. Simultaneous determination of angiotensins II and 17 by capillary zone electrophoresis in plasma and urine from hypertensive rats. Talanta 80:
17021712, 2010.
136. Tikellis C, Bialkowski K, Pete J, Sheehy K, Su Q, Johnston C,
Cooper M, Thomas M. ACE2 deficiency modifies renoprotection afforded by ACE inhibition in experimental diabetes. Diabetes 57: 1018
1025, 2008.
137. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ.
A human homolog of angiotensin-converting enzyme. Cloning and
functional expression as a captopril-insensitive carboxypeptidase. J Biol
Chem 275: 33238 33243, 2000.
138. Urata H, Kinoshita A, Misono KS, Bumpus FM, Husain A. Identification of a highly specific chymase as the major angiotensin II-forming
enzyme in the human heart. J Biol Chem 265: 22348 22357, 1990.
139. Van den Heuvel M, Batenburg WW, Jainandunsing S, Garrelds IM,
van Gool JM, Feelders RA, van den Meiracker AH, Danser AH.
Urinary renin, but not angiotensinogen or aldosterone, reflects the renal
renin-angiotensin-aldosterone system activity and the efficacy of reninangiotensin-aldosterone system blockade in the kidney. J Hypertens 29:
21472155, 2011.
140. Van Kats JP, Schalekamp MA, Verdouw PD, Duncker DJ, Danser
AH. Intrarenal angiotensin II: interstitial and cellular levels and site of
production. Kidney Int 60: 23112317, 2001.

141. Vidotti DB, Casarini DE, Cristovam PC, Leite CA, Schor N, Boim
MA. High glucose concentration stimulates intracellular renin activity
and angiotensin II generation in rat mesangial cells. Am J Physiol Renal
Physiol 286: F1039 F1045, 2004.
142. Wanka H, Kessler N, Ellmer J, Endlich N, Peters BS, Clausmeyer S,
Peters J. Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts. J Cell Mol Med 13: 2926 2937,
2009.
143. Westwood BM, Chappell MC. Divergent pathways for the angiotensin(112) metabolism in the rat circulation and kidney. Peptides 35: 190
195, 2012.
144. Wilson BA, Cruz-Diaz N, Marshall AC, Pirro NT, Su Y, Gwathmey
TM, Rose JC, Chappell MC. An angiotensin-(17) peptidase in the
kidney cortex, proximal tubules, and human HK-2 epithelial cells that is
distinct from insulin-degrading enzyme. Am J Physiol Renal Physiol 308:
F594 F601, 2015.
145. Wilson BA, Marshall AC, Alzayadneh EM, Chappell MC. The ins
and outs of angiotensin processing within the kidney. Am J Physiol Regul
Integr Comp Physiol 307: R487R489, 2014.
146. Wintroub BU, Schechter NB, Lazarus GS, Kaempfer CE, Schwartz
LB. Angiotensin I conversion by human rat chymotryptic proteinases. J
Invest Dermatol 83: 336 339, 1984.
147. Wysocki J, Ye M, Batlle D. Plasma and kidney angiotensin peptides:
importance of the aminopeptidase A/angiotensin III axis. Am J Hypertens
28: 1418 1426, 2015.
148. Yamaleyeva LM, Gilliam-Davis S, Almeida I, Brosnihan KB,
Lindsey SH, Chappell MC. Differential regulation of circulating and
renal ACE2 and ACE in hypertensive mRen2. Lewis rats with
early-onset diabetes. Am J Physiol Renal Physiol 302: F1374 F1384,
2012.
149. Yang R, Smolders I, Vanderheyden P, Demaegdt H, Van Eeckhaut
A, Vauquelin G, Lukazuk A, Tourwe D, Chai SY, Albiston AL,
Nahmias C, Walther T, Dupont AG. Pressor and renal hemodynamic
effects of the novel angiotensin A peptide are AT1a-receptor dependent.
Hypertension 57: 956 964, 2011.
150. Yang R, Walther T, Gembardt F, Smolders I, Vanderheyden P,
Albiston AL, Chai SY, Dupont AG. Renal vasoconstrictor and pressor
responses to angiotensin IV in mice are AT1a-receptor mediated. J
Hypertens 28: 487494, 2010.
151. Zhuo JL, Li XC. New insights and perspectives on intrarenal reninangiotensin system: focus on intracrine/intracellular angiotensin II. Peptides 32: 15511565, 2011.
152. Zisman LS, Keller RS, Weaver B, Lin Q, Speth R, Bristow MR,
Canver CC. Increased angiotensin-(17)-forming activity in failing
human heart ventricles: evidence for upregulation of the angiotensinconverting enzyme homologue ACE2. Circulation 108: 17071712,
2003.

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00618.2015 www.ajpheart.org