Advances in Biological Chemistry, 2011, 1, 93-102
doi:10.4236/abc.2011.13011 Published Online November 2011 (http://www.SciRP.org/journal/abc/
ABC
).
Published Online November 2011 in SciRes. http://www.scirp.org/journal/ABC
Garlic (Allium sativum) modulates the expression of
angiotensin II AT2 receptor in adrenal and renal tissues of
streptozotocin-induced diabetic rats
Mohamed H. Mansour*, Khaled Al-Qa tta n, Marth a Thomson, Musli m Ali
Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait City, Kuwait.
Received 14 September 2011; revised 19 October 2011; accepted 27 October 2011.
ABSTRACT
The loss of balance between the antagonistic activities
of angiotensin II AT1/AT2 receptors has been impli-
cated as a major mediator in the development of hy-
pertension and progressive nephropathy in experi-
mental diabetes. The present study was designed to
investigate the potential of garlic to modulate the
level of expression of the AT2 receptor in the adrenal
and renal tissues of diabetic rats. Three groups of
rats were studied after 8 weeks following diabetes
induction: normal, streptozotocin-induced diabetic
(control diabetic), and garlic-treated diabetic rats. A
polyclonal antibody of proven specificity to the AT2
receptor, as verified by Western blotting and emplo-
yed in immunohistochemical assays, indicated that
compared to normal rats, the highest adrenocortical
AT2 receptor expression was significantly shifted from
the zona glomerulosa to the zona fasciculate/ reticu-
laris, and was significantly reduced in adrenomedul-
lary chromaffin cells of control diabetic rats. In the
kidney, STZ treatments were associated with a signi-
ficant decrease in AT2 receptor expression throughout
glomeruli and all cortical and medullary tubular
segments. Compared to control diabetic rats, the la-
beling of the AT2 receptor in the garlic-treated dia-
betic group was restored among adrenocortical zona
glomerulosa cells and adrenomedullary chromaffin
cells and significantly reduced in the zona fasiculata,
and was also restored in glomeruli and throughout
renal cortical and medullary tubular segments, to le-
vels comparable to those observed in normal rats.
The capacity of garlic to modulate diabetes-induced
AT2 receptor down-regulation may be implicated in
restoring the recuperative processes mediated by AT2
receptors, which interfere with the development of
hypertensi on and nephropathy.
Keywords: AT 2 Receptor; Garlic; Streptozotocin-Induced
Diabetes; Immunohistochemistry
1. INTRODUCTION
The renin-angiotensin-aldosterone system (RAAS) is a
major regulator of blood pressure and sodium and water
homeostasis [1]. The octapeptide angiotensin II (Ang II)
is the primary mediator of the RAAS effects. Ang II in-
duces its effects by binding to two major receptor types,
AT1 and AT2 [2]. The AT1 receptors are ubiquitously ex-
pressed in adult tissues and mediate Ang II-induced
vasoconstriction, sodium reabsorption, aldosterone se-
cretion, and cell growth and proliferation [3-5]. The AT2
receptors are expressed abundantly during fetal devel-
opment, decline after birth and become minimally rep-
resented, in comparison with AT1 receptors, in various
adult tissues [6]. In these tissues, AT2 receptors target
Ang II-induced vasodilatation, apoptosis, antiprolifera-
tion, natriuresis and fluid/sodium homeostasis [7-9] and,
therefore, can be considered as functional antagonists to
the AT1 receptors [10]. Compelling evidence suggest that
the alteration in the expression and function of either
Ang II receptor type may be the site of regulation that
affects the initiation and progression of tissue remodel-
ing in pathophysiological conditions [11-12].
As manifested in diabetic patients and animal models,
the hyperglycemia-induced AT1 receptor up-regulation in
the adrenal, kidney and other tissues mediates the in-
crease of Ang II local effects in stimulating aldosterone
production and over-activating renal sodium transporters
causing sodium retention and hypertension [12-14].
Within the kidney, the up-regulated AT1 receptor signal-
ing increases vascular resistance, glomerular capillary
pressure and mechanical stretch-induced glomerular
injury, and stimulates production of reactive oxygen
species and extracellular matrix in the mesangium and
tubulo-interstitium [2,14-16], which collectively perpe-
trates the development of diabetic nephropathy. Interest-
ingly, early diabetes exerts an opposite influence on AT2
receptor and significantly decreases its expression in
glomeruli and tubular segments of the kidney [17]. The

M. H. Mansour et al. / Advances in Biological Chemistry 1 (2011) 93-102 94
down-regulation of intrarenal AT2 receptor expression
implies the subsequent decrease in AT2-mediated me-
chanisms that may oppose the detrimental effects of AT1
receptor signaling, including inhibition of cell growth
[18,19] as well as NO/cGMP-dependent vasodilatation
and inhibition of renal sodium transporters [20-22]. This
could result in an amplification of AT1-mediated effects
on vasoconstriction, glomerulosclerosis or cell hyper-
trophy, and thus contribute further to progressive injury
in diabetic nephropathy [17,23].
Current views suggest that pharmacologically active
blockers of the RAAS may be beneficial in the man-
agement of diabetic complications including hyperten-
sion and nephropathy [24,25]. Angiotensin receptor block-
ers and angiotensin-converting enzyme inhibitors are
used to treat hypertension and to improve renal function
in diabetes [26]. Based on the suggestion that a balance
between AT1- and AT2-receptor-mediated cell-signaling
events may be a determinant of progression rate in dia-
betic nephropathy [17], a debate of preferential use of
angiotensin receptor blockers over angiotensin-conver-
ting enzyme inhibitors have been initiated. The basis of
such preference lies in that angiotensin-converting en-
zyme inhibitors lower Ang II production, leading to re-
duction in the functions of AT1 and AT2 receptors,
whereas angiotensin receptor blockers selectively block
AT1 receptors and leave the AT2 receptors unopposed to
function [26]. Apparently, restoration of AT2 receptor
activity, in early diabetes, may add more therapeutic
efficacy in protecting against the development of renal
morphologic and functional changes seen during the pro-
gression of hypertension and nephropathy.
Currently, the reliance on natural products is gaining
popularity in combating various physiological threats,
including diabetic complications, associated with the
dysregulation of the RAAS [27]. In experimental diabe-
tes, garlic, used either as a whole raw extract or as iso-
lated organosulphur constituents, is one of the best stud-
ied herbal remedies with documented anti-diabetic ac-
tivities [28-32]. These activities include lowering serum
glucose and cholesterol levels [30,31,33], inhibiting Ang
II and promoting vasodilatation [29], preventing adrenal
hypertrophy [34], attenuating oxidative stress [35], in-
hibiting the formation of advanced glycation endpro-
ducts [36], ameliorating hypertension and delaying the
progression of diabetic nephropathy [31,32,35,36]. No-
netheless, the potential of garlic in targeting the level of
expression of Ang II receptors is, as yet, not fully ex-
plored. In a recent study, we reported on the efficacy of
an aqueous extract of raw garlic in modulating the up-
regulated expression of Ang II AT1 receptors in the ad-
renal and renal tissues in early diabetes [37]. In the pre-
sent study, immunohistochemical evidence is presented
for the capacity of garlic treatments to modulate the
down-regulated expression of Ang II AT2 receptors in
adrenal and renal tissues of streptozotocin-induced dia-
betic rats.
2. MATERIALS AND METHODS
2.1. Antibodies and Reagents
Except where noted, all chemicals were reagent grade
and purchased from Sigma Chemical Company (St.
Louis, MO, USA). Polyclonal anti-AT2 antibody (sc-
7421, rabbit IgG specific to an epitope mapping within
the N-terminal extracellular domain of the human AT2
polypeptide) and peroxidase-conjugated goat anti-rabbit
IgG antibody were purchased from Santa Cruz Biotech-
nology, Inc. (Santa Cruz, Ca, USA). Gel electrophoresis
reagents, peroxidase-conjugated molecular weight stan-
dards and nitrocellulose membranes (0.45 ) were ob-
tained from BioRad (Richmond, CA, USA). An eight
amino acid peptide, corresponding to amino acids 10 -
17 (Thr-Ser-Arg-Asn-Ile-Thr-Ser-Ser) of the first extra-
cellular domain, as deduced from the published AT2 re-
ceptor cDNA sequence [38], was synthesized manually
on the base-labile linker 4-(hydroxymethyl)-benzoyloxy-
methyl and supplied by The Protein/DNA Technology
Center (The Rockefeller University, NY, USA). The oc-
tapeptide was conjugated to bovine serum albumin (BSA)
and coupled to CNBr-activated Sepharose 4B as descri-
bed previously [39]. An aqueous garlic extract (500 mg/
ml) was prepared from locally purchased, peeled garlic
cloves by homogenization in cold, sterile 0.9% NaCl for
12 min followed by filtration and centrifugation at 200 g
for 10 min to remove insolubles as previously described
[30].
2.2. Animals
Adult male Sprague-Dawley (SD) rats (England) weigh-
ing 150 - 200 gm were bred and raised at the animal
house of the Department of Biological Sciences, Kuwait
University, and used in the present investigation. Rats
were given standard laboratory chow (170 mMol Na+/kg)
and water ad libitum and kept under standard conditions
(23˚C ± 2˚C, 12 h light, 12 h darkness). Diabetic rats,
induced by injecting streptozotocin (STZ, 60 mg/kg)
intraperitoneally into overnight fasting rats, were deter-
mined to be diabetic if they had elevated plasma glucose
concentrations ≥ 300 mg/dL five days post-injection, as
described previously [40]. STZ-induced diabetic rats
were divided into two groups (n = 8): group 1, the con-
trol diabetic group, received daily intra-peritoneal injec-
tions with saline, and group 2, the garlic-treated group,
received daily intraperitoneal injections with 500 mg/kg
of the garlic extract. All experiments were carried out at
8 weeks after diabetes induction. All animal procedures
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M. H. Mansour et al. / Advances in Biological Chemistry 1 (2011) 93-102 95
were performed according to the guidelines for animal
experimentation of Kuwait University, Faculty of Sci-
ence.
2.3. Solubilization of Cell-Membranes and
Endoglycosidase Tr eatments
Left adrenal glands and kidneys, excised from anesthe-
tized normal rats, were individually homogenized, solu-
bilized and extracted in 10 mM Tris/HCl, pH 8.0 con-
taining 2 mM phenylmethylsulfonyl fluoride and 2%
deoxycholate by an automatic homogenizer followed by
sonic disruption and stirring at room temperature for 2 h
and three cycles of freezing at –20˚C and thawing at
room temperature. Solubilized cell-membrane lysates
were recovered in supernatants, following centrifugation
at 100.000 g for 1 h, and their protein content deter-
mined [41] using bovine serum albumin (BSA) in the
same buffer as a standard. Cell-membrane lysates of
both organs (120 μg protein) were separately precipi-
tated with 20% trichloroacetic acid and ice-cold acetone
for 1 h at –20˚C, washed for 1 h with acetone at –20˚C
and reconstituted in 50 μl of 100 mM sodium phosphate,
pH 6.1, 50 mM EDTA, 1% Nonidet P-40 containing 20
mU of Endo-F (Endo-ß-N-acetylglucosaminidase F, from
Flavobacterium meningosepticum, 600 U/mg, Sigma
Chem. Comp., St. Louis, MO). Samples were incubated
for 18 h at 37˚C, precipitated with equal volume of 20%
trichloroacetic acid, washed with cold acetone and dried
under nitrogen gas before analysis by polyacrylamide
gel electrophoresis. Control samples were similarly trea-
ted but in the absence of Endo-F.
2.4. We s tern Blotting
Aliquots of solubilized cell-membrane lysates (120 g
protein) collected from adrenal and/or kidney tissues of
normal SD rats, and either kept untreated or treated with
Endo-F, were individually precipitated, reconstituted in
sample buffer and resolved with 10% resolving gels,
under non-reducing or reducing conditions, by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SD-
S-PAGE) as described by Laemmli [42]. Resolved pro-
tein samples were electrophoretically transblotted to
0.45  nitrocellulose membranes at 100 V (constant
voltage) for 1.5 h at 4˚C in electrophoretic transfer buffer,
pH 8.3 using a Bio Rad Mini Trans-Blot electrophoretic
transfer cell. Nitrocellulose membranes were washed 3
times with 200 mM PBS, pH 7.2 containing 0.05%
Tween 40 (PBS-Tween buffer), each for 15 min with
constant agitation and nonspecific binding sites blocked
by incubation for 1 h in blocking buffer (3% BSA in
PBS-Tween buffer, pH 7.2). Membranes were then
washed thrice in PBS-Tween buffer, pH 7.2 and subse-
quently probed by the anti-AT2 receptor antibody (di-
luted to 1:500 in PBS-Tween buffer, pH 7.2) by incuba-
tion for 2 h at room temperature and then overnight at
4˚C with constant agitation. Control blots were prepared
by substituting the specific antibody with an equal vol-
ume of the anti-AT2 antibody preabsorbed with an AT2
octapeptide/BSA complex-coated CNBr-activated Sep-
harose 4B beads. Following the incubation time, mem-
branes were washed thrice in PBS-Tween buffer, pH 7.2,
treated for 1 h at room temperature with peroxidase-con-
jugated goat anti-rabbit IgG antibody (diluted to 1:1000 in
PBS-Tween buffer, pH 7.2) and the reactions visualized
by treatments with metal-enhanced 3,3-diaminobenzidine
(DAB) tablets reconstituted in distilled water. Mem-
branes were allowed to air-dry, photographed and rela-
tive molecular weights estimated using peroxidase-con-
jugated Bio Rad broad range molecular weight standards,
which were analyzed under identical conditions in par-
allel to the protein samples.
2.5. Preparation of Tissue Sections
Left adrenal glands and kidneys of anesthetized normal,
control diabetic and/or garlic-treated diabetic rats were
individually excised and placed in vials containing 3 ml
of Bouin’s fixative for 24 h - 48 h at room temperature.
The tissues were carried through a routine paraffin em-
bedding technique, which included dehydration through
a series of ethanol concentrations 50%, 70%, 90% and
100%, clearing in toluene, embedding in paraffin wax,
and finally sectioning of the paraffin blocks into 3 m -
4 m thick sections on a rotary microtome. The sections
were picked up on clean slides after spreading them in a
water bath at 40˚C. The slides were air-dried to be used
for subsequent staining.
2.6. Labeling of Tissue Sections with
Anti-AT2 Antibody
Tissue sections were examined for AT2 receptor distribu-
tion by an indirect immunohistochemical labeling tech-
nique. Tissue sections were de-waxed in xylene, hy-
drated with a series of 90%, 75% and 60% ethanol and
washed with PBS, pH 7.2. Sections were quenched by
the addition of 10% normal goat serum and 0.3% hy-
drogen peroxide in PBS, pH 7.2 for 1 h and then indi-
vidually labeled for 45 min in a humidified chamber
with 300 l of the anti-AT2 receptor antibody (diluted to
1:100 with PBS, pH, 7.2). After several washes with 200
l PBS, pH 7.2, the sections were incubated for 45 min
with 300 l of peroxidase-conjugated goat anti-rabbit
IgG antibody (diluted to 1:200 in PBS, pH 7.2), followed
by a 10-min treatment with 400 l of fast DAB recon-
stituted in water. All sections were counter stained with
100 l haematoxylin (Gill #1) for 1 min and examined
by light microscopy for positive labeling of cells ex-
pressing AT2 receptors. Control sections were identically
stained by replacing the specific antibody with anti-AT2
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M. H. Mansour et al. / Advances in Biological Chemistry 1 (2011) 93-102 96
antibody preabsorbed with an AT2 octapeptide/ BSA
complex-coated CNBr-activated Sepharose 4B beads. Mi-
crophotographs were taken using Olympus AH-3 auto-
mated microscope (Tokyo, Japan), equipped with an
Olympus Vanox camera. Slides were quantitatively ex-
amined by using the Image-Pro Plus 5.1 software pro-
gram (Media Cybernetics, Silver Spring, MD). Each s-
lide was analyzed for AT2 receptor labeling in the adre-
nal cortex and medulla, and in the renal cortex and outer
and inner medulla, with three separate fields viewed in
each region and four independent samples for each
group. Data were expressed as mean values ±SE and
statistically analyzed using SPSS, Version 17. Groups
were compared with one-way ANOVA and p < 0.05 was
considered to be significant.
3. RESULTS
3.1. Characterization of the AT2 Receptor
Expressed in Adrenal and Renal Tissues
A polyclonal anti-AT2 receptor antibody was utilized (at
a dilution of 1:500) in probing whole adrenal gland and
kidney solubilized proteins (120 μg) of normal rats,
which were either untreated or treated with Endo-F and
resolved by SDS-PAGE. As judged by Western blotting
conducted in the absence of Endo-F treatments, the reac-
tivity of the polyclonal anti-AT2 receptor antibody was
selectively targeted towards 71.3 kDa and 66.8 kDa
components in both adrenal gland and kidney lysates
(Figure 1), analyzed under either reducing or non-re-
ducing conditions.
Following mild treatments with 20 mU of Endo-F and
analyses by Western blotting, a major 41 kDa band ap-
peared in addition to the original 71.3 kDa and 66.8 kDa
components in the lysates of both organs. It is notewor-
thy that none of these components were observed in
Western blots of either organ analyzed under identical
conditions but probed by anti-AT2 antibody preabsorbed
with an AT2 octapeptide/BSA complex-coated CNBr-
activated Sepharose 4B beads.
3.2. AT2 Receptor Expression in the Adrenal
The expression and zonal distribution of the AT2 receptor
was immunohistochemically investigated and compared
in the adrenal gland of normal, control diabetic and gar-
lic-treated diabetic rats by using a standard indirect la-
beling technique of de-waxed tissue sections. These sec-
tions were treated with aliquots of the polyclonal
anti-AT2 receptor antibody and counter-stained with he-
matoxylin. Control sections, stained with anti-AT2 anti-
body preabsorbed with an AT2 octapeptide/BSA com-
plex-coated CNBr– activated Sepharose 4B beads, were
investigated in parallel to allow comparisons of selective
tissue labeling. Among adrenal cortical zones in normal
Figure 1. Western blot of SDS-PAGE analysis of the AT2
receptor expressed in adrenal and renal tissues of SD rats.
Whole adrenal gland and kidney solubilized proteins (120
μg) of normal rats were either untreated (–) or treated (+)
with Endo-F, resolved by SDS-PAGE, blotted and probed
by a polyclonal anti-AT2 receptor antibody. Positions of
Bio Rad molecular weight (Mr × 10–3) standards and the
estimated molecular weights of the untreated and Endo-
F-treated AT2 receptor are indicated.
rats, light microscopy revealed that moderate labeling
with the anti-AT2 antibody was selectively evident among
virtually all cells in the zona glomerulosa, as well as few
scattered cells within the zona fasiculata and the zona
reticularis, which were otherwise uniformly marked by
their entire lack of labeling (Figure 2(a) and Figure
3(a)). In striking contrast, no immunohistochemical
staining for the AT2 receptor was detectable in the zona
glomerulosa, but was selectively notable among most
cells constituting the deep zona fasiculata as well as the
zona reticularis in control diabetic rats (Figure 2(b) and
Figure 3(b)). In garlic-treated diabetic rats, AT2 receptor
labeling with moderate intensity was restored among
cells of the zona glomerulosa, and was notable in the
zona reticularis and the outer region of the zona fasicu-
lata, but particularly faded among most of the deep re-
gion of the zona fasiculata (Figure 2(c) and Figure 3(c)).
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M. H. Mansour et al. / Advances in Biological Chemistry 1 (2011) 93-102 97
Similarly, distinct labeling patterns of the AT2 receptor
were observed in the adrenal medulla of the three rat
groups. As shown in Figure 3, labeling with high to
moderate intensity was confined to few chromaffin cells
in both normal (Figure 3(a)) and garlic-treated diabetic
(Figure 3(c)) rats, but involved more frequent chromaf-
fin cells in normal rats, and was almost absent in all
chromaffin cells of control diabetic rats (Figure 3(b)).
None of these specific cortical and medullary labeling
patterns were observed with adrenal sections treated
Figure 2. AT2 receptor expression in the adrenal cortex of
normal (A), control diabetic (B) and garlic-treated diabetic (C)
rats. (ca) capsule, (G) zona glomerulosa, (F) zona fasciculata.
No specific labeling was observed in adrenal tissue sections
labeled with the anti-AT2 antibody preabsorbed with an AT2
octapeptide/BSA complex (D). Bar = 200 µm, ×200.
Figure 3. AT2 receptor expression in the adrenal cortico-me-
dullary region of normal (A), control diabetic (B) and gar-
lic-treated diabetic (C) rats, (R) zona reticularis, (M) medulla.
No specific labeling was observed in adrenal tissue sections
labeled with the anti-AT2 antibody preabsorbed with an AT2
octapeptide/BSA complex (D). Bar = 200 µm, ×200.
with anti-AT2 antibody preabsorbed with an AT2 octape-
ptide/BSA complex-coated CNBr– activated Sepharose
4B beads (Figure 2(d) and Figure 3(d)). As depicted in
Table 1, quantification of AT2 receptor staining by com-
puter-based image analysis revealed a significant in-
crease in the expression of AT2 receptors in the adrenal
cortex of control diabetic rats compared with the normal
group. The adrenal cortex of garlic-treated diabetic rats
was associated with a significant decrease of AT2 recep-
tor immunostaining compared with control diabetic rats,
and comparable to the level observed in the normal
group. On the other hand, a significant reduction in AT2
receptor labeling was observed in the adrenal medulla of
control diabetic rats compared with the normal and gar-
lic-treated diabetic rats.
3.3. AT2 Receptor Expression in the Kidney
In immunohistochemical labeling experiments of kidney
sections of the three rat groups, light microscopy re-
vealed that variable patterns of AT2 receptor labeling
were associated with the cortex, the inner stripe of the
outer medulla as well as the inner medulla. As depicted
in Figure 4(a), cortical nephron segments of normal rats
exhibited AT2 receptor labeling of marked intensity in
glomerular endothelial cells and the entire epithelial lin-
ing of the distal convoluted tubules, but was uniformly
diffused in the epithelial lining of the proximal convo-
luted tubules. In control diabetic rats, AT2 receptor la-
beling was markedly reduced in glomerular endothelial
cells, appeared as faded patches at the basolateral side of
some of the epithelial cells lining the proximal convo-
luted tubules, and was evidently lacking among the
epithelial lining of the distal convoluted tubules (Figure
4(b)). On the other hand, except for the persistent lack of
AT2 receptor labeling in the epithelial lining of the distal
convoluted tubules, specific labeling with marked inten-
sity was observed in glomerular endothelial cells and the
Table 1. AT2 receptor quantification.
Normal
rats
Control
diabetic rats
Garlic-treated
diabetic rats
Adrenal
cortex 2.671 ± 752 5.890 ± 635* 3.114 ± 110
medulla 7.221 ± 369 1.265 ± 223* 5.967 ± 776
Kidney
cortex 5.221 ± 245 1.523 ± 447* 12.204 ± 545†
outer
medulla 12.567 ± 487 3.054 ± 325* 13.206 ± 715
Inner
medulla 6.321 ± 287 2.112 ± 101* 5.414 ± 614
Values are means ± SE (n = 4). Each value represents the number of pixels
that exceeded an arbitrary staining threshold per 300 × 300-pixel area. *P <
0.05 vs. Garlic-treated diabetic rats and normal rats. †P < 0.05 vs. normal rats.
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M. H. Mansour et al. / Advances in Biological Chemistry 1 (2011) 93-102 98
entire epithelial lining of the proximal convoluted tu-
bules in garlic-treated diabetic rats (Figure 4(c)). As in
the cortical regions, distinct labeling patterns of the AT2
receptor were also observed in the inner stripe of the
outer medulla of the three rat groups. In normal rats
(Figure 5(a)), binding of the anti-AT2 receptor antibody
was evident in the basolateral side of collecting ducts
and ascending Henle’s loop segments and, to a lesser
extent, the basolateral side of collecting tubules, without
any obvious involvement of interstitial cells outlining
the vasa recta bundles or the epithelial elements of de-
scending Henle’s loop segments. Compared to normal
rats, AT2 receptor labeling in control diabetic rats was of
significantly reduced intensity in the epithelia of col-
lecting ducts and tubules and ascending Henle’s loop
segments, and was completely lacking in interstitial cells
outlining the vasa recta bundles and the epithelia of de-
scending Henle’s loop segments (Figure 5(b)). As
shown in Figure 5(c), AT2 receptor labeling was accen-
tuated in the cytoplasm of the entire epithelial lining of
collecting ducts and ascending Henle’s loop segments
and was apparently detectable in interstitial cells outlin-
ing the vasa recta bundles, but was absent in collecting
tubules and descending Henle’s loop epithelia in gar-
lic-treated diabetic rats. In the inner medulla of control
and garlic-treated diabetic rats, AT2 receptor immu-
nostaining was localized to the apical membranes and
cytoplasm of inner medullary collecting duct cells (Fig-
ures 6(a) and (c)), whereas in diabetic rats the labeling
of these segments was markedly decreased (Figure 6(b)).
Figure 4. AT2 receptor expression in the renal cortex of normal
(A), control diabetic (B) and garlic-treated diabetic (C) rats.
(DCT) distal convoluted tubule, (G) glomerulus, (PCT) proxi-
mal convoluted tubule. No specific labeling was observed in
kidney tissue sections labeled with the anti- AT2 antibody pre-
absorbed with an AT2 octapeptide/BSA complex (D). Bar =
100 µm, ×100.
None of these specific cortical and medullary labeling
patterns were observed with renal sections treated with
anti- AT2 antibody preabsorbed with an AT2 octapep-
tide/BSA complex-coated CNBr– activated Sepharose
4B beads (Figure 4(d), Figure 5(d) and Figure 6(d)).
Quantification of AT2 receptor staining by computer-
based image analysis revealed a significant decrease in
AT2 receptor labeling in the renal cortical and medullary
regions of the control diabetic group compared with
normal rats. Medullary regions of garlic-treated diabetic
Figure 5. AT2 receptor expression in the outer medulla of the
kidney of normal (A), control diabetic (B) and garlic-treated
diabetic (C) rats. (A) ascending limb of Henle’s loop, (CD)
collecting duct, (CT) collecting tubule, (D) descending limb of
Henle’s loop, (VR) vasa recta bundle. No specific labeling was
observed in kidney tissue sections labeled with the anti-AT2
antibody preabsorbed with an AT2 octapeptide/BSA complex
(D). Bar = 100 µm, ×100.
Figure 6. AT2 receptor expression in the inner medulla of the
kidney of normal (A), control diabetic (B) and garlic-treated
diabetic (C) rats. (IMCT) inner medullary collecting duct. No
specific labeling was observed in kidney tissue sections labeled
with the anti-AT2 antibody preabsorbed with an AT2 octapep-
tide/BSA complex (D). Bar = 100 µm, ×100.
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rats were marked by levels of AT2 receptor labeling that
were significantly higher than the control diabetic group
and comparable to those observed in normal rats. In the
cortical region, AT2 receptor labeling of garlic-treated
diabetic rats was significantly higher than the normal
and control diabetic groups (Table 1).
4. DISCUSSION
Garlic has a wide range of therapeutic applications. In
experimental diabetes, sulfur-containing and non-sulfur
constituents of garlic work synergistically to exhibit an-
tithrombotic, antioxidant, hypocholesterolaemic, hypo-
glycaemic as well as hypotensive potentials, which col-
lectively ameliorate the development and progression of
diabetic complications, including nephropathy and hy-
pertension [28-36]. Nonetheless, neither the precise
mechanisms that underlie the anti-diabetic potentials of
garlic nor the nature of the key receptor system(s) tar-
geted, are fully understood. In hypertensive diabetic rats,
altered gene and protein expression of the Ang II AT1
and AT2 receptor types and the loss of balance between
their antagonistic activities, in renal and other tissues,
have been directly implicated in the development of
early changes associated with diabetic nephropathy [2,4,
5,12,14,17,21]. In a recent report, the remarkable capac-
ity of garlic treatments to modulate the upregulated ex-
pression of AT1 receptors, associated with hypersecretion
of aldosterone and the impairment of renal vascular and
tubular functions in early diabetes, has been demon-
strated [37]. The present study was designed to investi-
gate the level of expression of the AT2 receptor in the
adrenal and renal tissues of normal, control diabetic and
garlic-treated diabetic rats.
In the present study, AT2 receptor expression in adre-
nal and renal tissues was investigated using a polyclonal
anti-AT2 antibody of proven specificity to an epitope
mapping within the N-terminal extracellular domain of
the AT2 polypeptide [43]. In Western blots, this antibody
was selectively targeted towards 71.3 kDa and 66.8 kDa
components in both adrenal gland and kidney lysates,
which were in direct agreement with the estimated mo-
lecular weight of glycosylated AT2 receptors previously
detected in rat and other mammalian tissues [17,44,45].
Also consistent with the established presence of five
highly-conserved N-linked glycosylation sites along the
AT2 receptor sequence [45], mild enzymatic deglycosy-
lation treatments resulted in the detection of a lower
molecular weight component of 41 KDa in both organs,
which corresponded exactly to the predicted mass of the
deglycosylated protein back-bone deduced from the
cDNA sequence of the AT2 receptor [38,46]. In normal
rats, the binding of the anti-AT2 antibody was immuno-
histochemically demonstrable in the zona glomerulosa of
the adrenal cortex and scattered chromaffin cells of the
adrenal medulla and in glomeruli and proximal and dis-
tal convoluted tubules in the renal cortex, collecting tu-
bules and ducts and ascending Henle’s loop segments in
the inner stripe of the outer renal medulla, as well as
inner renal medullary collecting ducts. These selective
labeling patterns, observed in adrenal and renal tissues,
were all in accordance with AT2 receptor distribution in
both organs as previously determined by immunohisto-
chemical, in vitro autoradiographic localization and in
situ hybridization techniques [6,17,47,48]. None of the
glycosylated or deglycosylated forms of the receptor
observed in Western blots or the selective immunohisto-
chemical labeling patterns were detectable when the
antibody was preabsorbed with an AT2 octapeptide, cor-
responding to amino acids 10 - 17 of the first extracellu-
lar domain of the receptor polypeptide, thus establishing
the specificity of the polyclonal antibody in selectively
binding the AT2 receptor in adrenal and renal tissues.
Confirming the reported loss of balance in the levels
of AT1 and AT2 receptor expression in early diabetes [2,
4,5,12,14,17,21], STZ treatments in the present study
were paralleled by quantitative and qualitative altera-
tions in the pattern of AT2 receptor expression in both the
adrenal gland and the kidney. Compared to normal rats,
the highest adrenocortical AT2 receptor expression was
significantly shifted from the zona glomerulosa to the
zona fasciculate/reticularis, and was significantly re-
duced in adrenomedullary chromaffin cells of STZ-
treated, control diabetic rats. In this rat group, the reduc-
tion of AT2 receptor expression in the zona glomerulosa
was in direct contrast to the increase in AT1 receptor ex-
pression observed in this zone in an earlier report [37]
and may imply a decrease in AT2-mediated inhibition of
cell growth [18,19] and the amplification of AT1-medi-
ated effects on cell hypertrophy and aldosterone synthe-
sis and release [2-5], leading to electrolyte imbalance
and the development of hypertension. The significant
reduction of AT2 receptor expression in adrenomedullary
chromaffin cells may be also implicated in a possible
loss of the synergistic AT1/AT2 receptor cross-talk sug-
gested to regulate basal and stress-induced tyrosine hy-
droxylase transcription rates [49], and thereby leading to
dysregulation of adrenomedullary catecholamine secre-
tions. Interestingly, the up-regulatory shift in AT2 recep-
tor expression in the zona fasiculata/reticularis, which
was also observed earlier for AT1 receptor expression
[37], has been consistently observed with either receptor
type under various abnormal physiological and patho-
physiological conditions including dietary sodium re-
striction, sodium loading, stimulated levels of aldoster-
one or Ang II, and water deprivation [47,50-52]. Al-
though the exact functional significance of AT1/AT2 re-
ceptor perturbations in these zones is still to be eluci-
dated, their altered expression may represent a signifi-
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M. H. Mansour et al. / Advances in Biological Chemistry 1 (2011) 93-102 100
cant adaptive factor in adrenal tissue remodeling under
various pathological conditions, including early diabetes.
In the kidney, the present study also revealed that STZ
treatments were associated with a significant decrease in
AT2 receptor expression throughout all nephronal seg-
ments including glomeruli and proximal and distal con-
voluted tubules in the cortex, collecting tubules and
ducts and ascending Henle’s loop segments in the inner
stripe of the outer medulla, as well as inner medullary
collecting ducts. The observed decrease in total intrare-
nal expression of AT2 receptors is in direct accordance
with previous observations in the same model of ex-
perimental diabetes [17], and contrasts the documented
effects of STZ treatments in upregulating intrarenal ex-
pression of AT1 receptors [1,2,4,11,12,24,26,37]. Thus,
in early diabetes, the expected decline in AT2-mediated
mechanisms may potentiate the pathological augmenta-
tion of intrarenal AT1-mediated activities promoting so-
dium retention and hypertension, glomerulosclerosis and
proteinuria, tubulo-interstitial cell hypertrophy and hy-
perplasia associated with stimulation of extracellular
matrix production, which are the hallmark of diabetic
nephropathy [2,12-16].
A major observation of the present investigation was
the significant modulation of the AT2 receptor expressed
in adrenal and renal tissues of garlic-treated diabetic rats.
Compared to the control diabetic rats, AT2 receptor ex-
pression was restored among adrenocortical zona
glomerulosa cells and adrenomedullary chromaffin cells
and significantly reduced in the zona fasiculata of gar-
lic-treated diabetic rats. In the renal tissues of this rat
group, AT2 receptor expression was also significantly
restored in glomeruli and throughout renal cortical and
medullary tubular segments, to levels comparable to
those observed in normal rats. Along with the reported
efficacy of garlic treatments in reducing diabetes-in-
duced AT1 receptor up-regulation [37], the capacity of
garlic in modulating diabetes-induced AT2 receptor down-
regulation may imply not only reversing the detrimental
consequences of excessive AT1 receptor signaling, which
is pivotal in the dysregulation of adrenal secretions and
alterations in renal hemodynamic and tubular functions,
but also restoring the recuperative processes mediated by
AT2 receptors in both organs. This notion may be sup-
ported by the documented AT2-mediated activities in
inhibiting aldosterone hypersecretion and regulating
catecholamine levels in the adrenal gland [1,7-9,49],
inhibiting the sodium pump [53] and Na+-, K+-ATPase
activity [20-22] in renal proximal tubules thereby pro-
moting natriuresis/diuresis and hypotension, and inhib-
iting vasoconstriction and cell hypertrophy [7-9,18,19]
thereby interfering with excessive renal glomerular and
tubular remodeling. In this regard, our observations may
lend support to the documented efficacy of garlic treat-
ments in ameliorating diabetic complications in STZ-
treated rats [28-36] and further suggest that garlic may
alleviate diabetic-induced hypertension and nephropathy
by restoring the diabetic-induced loss of balance in
AT1/AT2 receptor expression in key target organs. Future
studies on the significance of constituent garlic metabo-
lites, acting individually or in concert, may clarify the
mechanisms that underlie its beneficial capacity in mo-
dulating the expression of selective receptor molecules
implicated in diabetes-associated disorders.
5. ACKNOWLEDGEMENTS
The reported work was supported by Research Project # 2007-1302-04,
funded by Kuwait Foundation for the Advancement of Science, and
Research Project # SL 06/08, funded by Kuwait University.
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