The beneficial effects of Allicin in chronic kidney disease are comparable to Losartan

Article abstract of DOI:10.3390/ijms18091980

Recent studies suggest that allicin may play a role in chronic kidney disease (CKD), reducing hypertension and oxidative stress and improving renal dysfunction. In the present study, CKD was induced by 5/6 nephrectomy and the animals were divided into four treatment groups as follows: control (C), CKD, CKD+allicin (40 mg/kg pathway oral) (CKDA), and CKD+Losartan (20 mg/kg) (CKDL). After CKD induction, the rats developed hypertension from week 3 to the end of the study. This was associated with increased creatinine and blood urea nitrogen (BUN) levels in serum, increased albuminuria, increased urinary excretion of N-acetyl-β-D-glucosaminidase (NAG), increased nephrin expression, and incrased histological alterations in the cortex. The levels of angiotensin receptors and endothelial nitric oxide synthase (eNOS) were decreased in the renal cortex from the CKD group. Otherwise, lipid and protein oxidation were higher in the CKD group than in the control group. A disturbance was observed in the expression levels of the nuclear factor erythroid 2-related factor 2/Kelch ECH associating protein 1 system (Nrf2/keap1) and the antioxidant enzymes catalase, superoxide dismutase, and heme oxygenase-1. Allicin or losartan treatments relieved renal dysfunction, hypertension, and oxidative stress. In addition, both treatments showed the same efficacy on the expression of angiotensin receptors, the nephrin, Nrf2/keap1 pathway, and eNOS. Further in silico analyses suggest that allicin and losartan could have a common mechanism involving interaction with AT1 receptors. Allicin showed antihypertensive, antioxidant, and nephroprotective effects. The beneficial effects showed by allicin are similar, or even better, than those of losartan. In fact, the effect of allicin on blood pressure and renal function is comparable to reductions seen with losartan, a prescription drug commonly used as a first-line therapy.

Full text of DOI:10.3390/ijms18091980

International Journal of
Molecular Sciences
Article
The Beneficial Effects of Allicin in Chronic Kidney
Disease Are Comparable to Losartan
1
1
1
Ehécatl Miguel Ángel García Trejo , Abraham Said Arellano Buendía , Omegar Sánchez Reyes ,
1
2
3
Fernando Enrique García Arroyo , Raúl Arguello García , María Lilia Loredo Mendoza ,
Edilia Tapia , Laura Gabriela Sánchez Lozada and Horacio Osorio Alonso *
1
1
1,
1
Renal Physiopathology Laboratory, Depterment of Nephrology, Instituto Nacional de Cardiología
“
Ignacio Chávez”, Mexico City 14080, Mexico; dr.ehecatl@hotmail.com (E.M.Á.G.T.);
neoabraham_7@hotmail.com (A.S.A.B.); tuzome_sanrey23@hotmail.com (O.S.R.);
jonibertojr@hotmail.com (F.E.G.A.); ediliatapia@hotmail.com (E.T.); lgsanchezlozada@gmail.com (L.G.S.L.)
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados-IPN,
Mexico City 07360, Mexico; raularguellogarcia@yahoo.com
Histopathology Laboratory, Research Subdivision, School of Medicine, Universidad Panamericana,
Donatello 43, Mexico City 03910, Mexico; lloredo@up.edu.mx
2
3
*
Correspondence: horacio.osorio@cardiologia.org.mx; Tel.: +52-55732911 (ext. 1263)
Received: 20 July 2017; Accepted: 13 September 2017; Published: 16 September 2017
Abstract: Recent studies suggest that allicin may play a role in chronic kidney disease (CKD),
reducing hypertension and oxidative stress and improving renal dysfunction. In the present study,
CKD was induced by 5/6 nephrectomy and the animals were divided into four treatment groups
as follows: control (C), CKD, CKD+allicin (40 mg/kg pathway oral) (CKDA), and CKD+Losartan
(
20 mg/kg) (CKDL). After CKD induction, the rats developed hypertension from week 3 to the
end of the study. This was associated with increased creatinine and blood urea nitrogen (BUN)
levels in serum, increased albuminuria, increased urinary excretion of N-acetyl- -D-glucosaminidase
NAG), increased nephrin expression, and incrased histological alterations in the cortex. The levels of
β
(
angiotensin receptors and endothelial nitric oxide synthase (eNOS) were decreased in the renal cortex
from the CKD group. Otherwise, lipid and protein oxidation were higher in the CKD group than in
the control group. A disturbance was observed in the expression levels of the nuclear factor erythroid
2
-related factor 2/Kelch ECH associating protein 1 system (Nrf2/keap1) and the antioxidant enzymes
catalase, superoxide dismutase, and heme oxygenase-1. Allicin or losartan treatments relieved renal
dysfunction, hypertension, and oxidative stress. In addition, both treatments showed the same
efficacy on the expression of angiotensin receptors, the nephrin, Nrf2/keap1 pathway, and eNOS.
Further in silico analyses suggest that allicin and losartan could have a common mechanism involving
interaction with AT1 receptors. Allicin showed antihypertensive, antioxidant, and nephroprotective
effects. The beneficial effects showed by allicin are similar, or even better, than those of losartan.
In fact, the effect of allicin on blood pressure and renal function is comparable to reductions seen with
losartan, a prescription drug commonly used as a first-line therapy.
Keywords: allicin; hypertension; chronic kidney disease; oxidative stress; losartan
1
. Introduction
Hypertension is the leading cause of chronic kidney disease (CKD) and end-stage renal disease
(
ESRD), contributing to the disease itself or, most commonly, contributing to its progression. Likewise,
hypertension is reported as a consequence of kidney injury [ ]. In the United States, approximately
0% of incident ESRD cases are attributed to hypertension [ ]. Hypertension is highly prevalent in
patients with CKD, playing a role in the high cardiovascular morbidity and mortality of this particular
1
2
3
Int. J. Mol. Sci. 2017, 18, 1980; doi:10.3390/ijms18091980
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 1980
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population. In fact, the risk of cardiovascular death in this patient group is greater than the risk of
progression to ESRD. Conversely, CKD is the most common form of secondary hypertension, and
mounting evidence suggests that it is an independent risk factor for cardiovascular morbidity and
mortality [3–5].
In CKD patients, the goal of hypertension management involves not only cardiovascular
protection by lowering blood pressure (BP) to the appropriate level but also slowing the progression of
kidney disease. The latter often includes the management of proteinuria, which is itself associated
with both the risk of cardiovascular disease and progression to ESRD [6,7]. Therefore, it is of great
importance to choose an appropriate antihypertensive agent for patients with CKD.
Current therapies include antihypertensive drugs such as angiotensin II-converting enzyme
inhibitors (ACEI) or angiotensin II receptor blockers (ARBs), both as first choice drugs.
These antihypertensives effectively reduce hypertension and cardiovascular diseases, providing
nephroprotection and thereby slowing the progression of CKD [7,8]. In addition, these particular
drugs have the ability to reduce cardiovascular morbidity and mortality.
In recent years, studies on the beneficial effects of extracts of Allium sativum L. (garlic) in the
treatment of chronic diseases such as diabetes and hypertension have been carried out [9–12]. The use
of garlic as an antihypertensive it is not well established, probably because the active substances
responsible for the therapeutic effects are not known with certainty. Among the active constituents in
garlic, one major component is allicin (thio-2-propene-1-sulfinic acid S-allyl ester), which is formed
from the stable precursor S-allyl cysteine-S-oxide (alliin) by the action of the enzyme alliinase when
garlic cloves are crushed or macerated [13]. Allicin has been shown to lower blood pressure in
hypertensive patients, as well as in hypertensive rats [14–17].
CKD places the patient into the highest cardiovascular risk level, irrespective of traditional
cardiovascular (CV) risk factor stratification. In this regard, we recently reported that allicin
treatment in rats with subtotal nephrectomy improves cardiac function and ameliorates oxidative
stress and hypertension [18], supporting the hypothesis that allicin could be important as a
complementary and/or alternative therapy to reduce the progression of CKD and decrease the risk of
cardiovascular disease.
To position our findings in line with translational research, our aim in the present study was
to compare the effects of allicin with losartan, a first-line and well-known antihypertensive drug,
in experimental CKD therapy with particular emphasis on kidney function and hypertension.
2
. Results
2
.1. Physiological Data
All experimental groups had a similar mean body weight at the beginning of the experiment.
Nevertheless, six weeks later, the body weight of the CKD group was lower compared to that of the
control group (Table 1). Interestingly treatment with allicin or losartan stimulated weight gain at higher
levels than the untreated group (Table 1), and weight differences were not found between losartan and
allicin treatment (Table 1).
Renal function was evaluated by the determination of creatinine and blood urea nitrogen (BUN)
levels in serum and creatinine clearance. As expected in this experimental model of CKD, the subtotal
nephrectomy resulted in a renal dysfunction status at six weeks of follow-up, which was evidenced by
the significant increase in creatinine and BUN levels in serum, as well as the reduction in creatinine
clearance (Table 1). The treatment with allicin or losartan showed beneficial effects. Thus, the body
weight and creatinine clearance were increased with both treatments with respect to the untreated
CKD rats. On the other hand, the creatinine and BUN levels in serum were decreased with allicin
or losartan when compared with the untreated group (Table 1). Treatments with allicin or losartan
showed the same efficacy on these parameters; however, losartan was slightly more efficient than
allicin in reducing creatinine levels in serum (Table 1).

Int. J. Mol. Sci. 2017, 18, 1980
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Table 1. The physiologic characteristics of experimental groups.
Control
CKD
30.81 *
1.43 ± 0.17 *
0.751 ± 0.09 *
63.16 ± 10.96 *
CKDA
CKDL
+
+
Body weight (g)
376.53 ± 2.38 331.69
±
364.45 ± 16.94
370.6 ± 6.05
,
+
,+,#
Serum creatinine (mg/dL)
Creatinine clearance (mL/min)
Blood urea nitrogen (mg/dL)
0.42 ± 0.02
1.42 ± 0.12
4.16 ± 0.40
1.122 ± 0.10 *
0.825 ± 0.28 *
,
+
,+,#
,+
1.05 ± 0.10 *
1.19 ± 0.09 *
,
+
42.18 ± 6.21 *
38.66 ± 3.77 *
CKD: chronic kidney disease; CKDA: chronic kidney disease, allicin-treated; CKDL: chronic kidney disease,
+
#
losartan-treated. Data represent the mean
versus CKDA.
±
SEM, n = 6. * p < 0.05 versus control; p < 0.05 versus CKD; p < 0.05
2
.2. Systolic Blood Pressure (SBP)
The progression of CKD is associated with high blood pressure, which may develop even at early
stages in the course of the disease, and has been associated with worsening renal function as well. Rats
with CKD induction developed high blood pressure, which was evident at three weeks of follow-up
and was severe at six weeks when compared with the control group (Figure 1). Three weeks after CKD
induction, there was an increase in the SBP in the CKD group by approximately 30% with respect to
the control group (Figure 1).
In the CKD allicin-treated group, there was a significant decrease ( 10 mmHg) in the SBP as
≈
compared to the untreated CKD group (Figure 1). Compared with losartan, the allicin treatment did
not show significant differences (Figure 1).
Figure 1. Systolic blood pressure at three and six weeks of follow-up. Control, C; chronic kidney disease,
CKD; chronic kidney disease, allicin-treated, CKDA; and chronic kidney disease, losartan-treated,
CKDL. Values represent mean ± SEM, n = 6. * p < 0.05 versus C; + p < 0.05 versus CKD.
At six weeks of follow-up, rats with CKD developed severe systemic hypertension compared
with the control group (183.8
±
2.62 versus 125.8
±
1.36 mmHg, respectively) (Figure 1). The allicin
treatment prevented the increase in SBP, causing it to end at 146 mmHg, almost 40 mmHg lower than
that of the untreated group (Figure 1). Despite the antihypertensive effects of allicin and losartan,
they did not achieve blood pressure levels similar to those recorded in the control group. Compared
with losartan, the effect of allicin treatment did not show statistically significant differences (Figure 1).
Therefore, losartan and allicin showed equal effectiveness in hypertension reduction.
2
.3. Markers of CKD Progression
Blood pressure control is associated with renal protection, thereby slowing CKD progression.
Amongst the most significant markers of CKD progression are albuminuria and tubular parameters
such as urinary excretion of N-acetyl- -D-glucosaminidase (NAG). Thus, to address the issue of blood
β
pressure control and the progression of CKD we assessed albuminuria and nephrin as markers of
glomerular injury and NAG excretion as an indicator of tubular damage.

Int. J. Mol. Sci. 2017, 18, 1980
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At three weeks of follow-up, the CKD group showed higher albuminuria compared to the control
group (Figure 2a). Albuminuria was aggravated at the end of the study. Additionally, the urinary
excretion of NAG showed a similar trend, which suggested a time-dependent CKD progression at the
glomerular and tubular levels (Figure 2b).
The allicin or losartan treatments showed renal protection at the glomerular and tubular levels
(Figure 2). Although the losartan and allicin treatments were equally effective, neither was able to
completely prevent albuminuria reaching those values observed in the control group (Figure 2).
Since our results showed that hypertension and albuminuria were severe at the end of study,
we evaluated the integrity of the glomerular filtration barrier (i.e., nephrin expression) at this time.
Nephrin gene expression was assessed by Western blot assays of the kidney cortex. As can be seen in
Figure 2c, nephrin expression was increased in the CKD group when compared with the control group
and the allicin or losartan treatments attenuated this effect.
C
CKD CKDA CKDL
Nephrin
β-Actin
0
.75
.60
.45
.30
.15
.00
*
0
0
0
+
+
0
0
(
a)
(b)
(c)
Figure 2. Analysis of markers of renal injury: (
-D-glucosaminidase (NAG); and ( ) semi-quantitative analysis of nephrin protein expression in
the kidney. Control (C); chronic kidney disease (CKD); CKD, allicin-treated (CKDA); and CKD,
a) Albuminuria; (b) urinary excretion of N-acetyl
β
c
losartan-treated (CKDL). Data are expressed as mean
versus CKD.
±
SEM, n = 6. * p < 0.05 versus C; + p < 0.05
2
.4. Histopathological Study
Hematoxylin and eosin (HE) staining revealed very important histological changes in the renal
cortex of animals from the CKD group in comparison to the unaltered architecture in the kidneys of
the control group. The glomerular changes at three weeks of follow-up were characterized by a size
increase, mesangial expansion and fibrosis, Bowman’s capsule adhesions, capillary occlusion, and
decreased cellularity (Figure 3a). At six weeks post-renal ablation, these alterations were augmented,
except for the glomerular size, which remained approximately the same. The tubular alterations at
three weeks of CKD induction consisted of lumen expansion, epithelium thinning, and hyaline casts.
The first two lesions were increased at six weeks of follow-up (Figure 3a). As shown in Figure 3a,
the glomerular and tubular histological changes at three and six weeks of follow-up were diminished
in both treated groups (CKDA and CKDL).

Int. J. Mol. Sci. 2017, 18, 1980
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(
a)
1
1
1
4,000
2,000
0,000
+
*
+
*
*
150,000
25,000
00,000
5,000
*
*
*
1
1
8
,000
,000
,000
,000
0
7
6
4
2
50,000
25,000
0
(
b)
(c)
Figure 3. Representative microphotographs of the kidney cortexes of animals subjected to subtotal
renal ablation for the induction of CKD at three weeks (upper images) and six weeks (lower images)
of follow-up (
a). Quantitative analysis of glomerular area (b); and tubular epithelial area (c) at six
weeks of follow-up. Controls (C) show no histological abnormalities. The chronic kidney disease group
(
(
CKD) shows glomerular (arrows) changes as mesangial expansion and fibrosis and shows tubular
arrow heads) lesions as lumen size augmentation, epithelial thinning, and the presence of hyaline
casts (*). The allicin-treated (CKDA) and losartan-treated (CKDL) groups’ images demonstrate a clear
amelioration of these glomerular and tubular changes. (HE, original magnifications 100 ). Data are
×
expressed as mean ± SEM, n = 6. * p < 0.05 versus C; + p < 0.05 versus CKD.
Additionally, at six weeks, we quantified the glomerular and tubular areas. Our results
demonstrated an increase in glomerular area in the CKD group compared with the control group
(Figure 3b). In contrast, the epithelial tubular area of the CKD group was decreased in comparison
to that of the control group (Figure 3c). The decrease of this parameter is indicative of atrophy of the
tubular epithelium. The allicin and the losartan treatments in CKD groups greatly attenuated these
histologic alterations (Figure 3b,c).
At six weeks of follow-up, we assessed the fibrosis by Sirius Red staining. The fibrosis assessment
was significantly increased in the cortex of the CKD group in comparison to that of the control group
(
* p < 0.05). There was less fibrosis in the group with CKDA and CKDL when compared with the
+
untreated group ( p < 0.05) (Figure 4).

Int. J. Mol. Sci. 2017, 18, 1980
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(
a)
6
000
000
000
000
000
000
*
5
+
*
4
+
*
3
2
1
0
(
b)
Figure 4. Evaluation of fibrosis. (
a
) Red Sirius stained representative photomicrographs. (
) Extense red areas
) treated animals respectively, depict a
very important decrease in the red stained areas in comparison with B. Arrows localized collagen in the
different images. (Original magnifications 200 ). ( ) Quantitative analysis. Control (C); chronic kidney
A) Control
shows collagen in red only in the Bowman capsule and in the vicinity of an artery; (
fibrosis) in the cortical interstitium; Alicin ( ) and losartan (
B
(
C
D
×
b
disease (CKD); CKD, allicin-treated (CKDA); and CKD, losartan-treated (CKDL). Data are expressed as
mean ± SEM, n = 6. * p < 0.05 versus C; + p < 0.05 versus CKD.
2
.5. Expression of Angiotensin II Receptors in Kidney Cortex
It has been reported that the renin-angiotensin system (RAS) is involved in CKD progression;
thus, we assessed the protein expression of AT1R and AT2R in the renal cortex using immunoblotting
assays. In the CKD group, the protein expression of both AT1R and AT2R was reduced in relation to
the control group (Figure 5). The allicin treatment prevented the decrease in AT1 expression observed
in the untreated CKD group (Figure 5a). However, allicin did not completely restore the levels of AT1R
expression. By contrast, there were no changes in the AT1R expression levels between losartan-treated
or untreated CKD groups, and even these were lower compared with the allicin treatment group
(Figure 5a). On the other hand, allicin treatment restored the AT2 expression levels in contrast to those
observed in the untreated CKD group (Figure 5b). Additionally, losartan treatment was able to prevent

Int. J. Mol. Sci. 2017, 18, 1980
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the downregulation of angiotensin II receptors in the renal cortex from the CKD group (Figure 5).
In this context, we did not find differences between the allicin and losartan treatments.
C
CKD CKDA CKDL
C
CKD CKDA CKDL
AT1R
AT2R
β-Actin
β-Actin
0
.30
0.30
0.25
0.20
0.15
0.10
0.05
0.00
+
+
0.25
0.20
0.15
0.10
0.05
0.00
*
+
#
*
*
*
(
a)
(b)
Figure 5. Chronic kidney disease dysregulated angiotensin II receptors in the renal cortex.
) Angiotensin II type 1 receptor (AT1R) and ( ) Angiotensin II type 2 receptor (AT2R). Representative
(a
b
immunoblot and quantitative analysis of relative protein expression. Control, C; chronic kidney disease,
CKD; chronic kidney disease, allicin-treated, CKDA; chronic kidney disease, losartan-treated, CKDL.
Values are means
±
SEM of n = 6. * p < 0.05 versus C; + p < 0.05 versus CKD; # p < 0.05 versus CKDA.
2
.6. Evaluation of Renal Oxidative Stress
In CKD, there is an increase in pro-oxidant molecules and/or a decrease of antioxidant enzymes.
Hence, we studied the oxidative stress using lipid and protein oxidation as markers of oxidative stress
and genes under the control of the antioxidant Nrf2/Keap1 system.
In the cortex and medulla from rats with CKD there was an oxidative stress status, as demonstrated
by the content of oxidized proteins and lipid peroxidation, which was higher in the CKD group than
in the control animals. These results were observed in the cortex as well as in the medulla (Table 2).
Allicin treatment was as effective as losartan treatment in reducing the lipid oxidation in the cortex and
medulla (Table 2). Concerning the effects of allicin and losartan on oxidized proteins, both treatments
reduced the oxidation of proteins in the cortex (Table 2). This effect was not observed in the renal
medulla, where only allicin showed antioxidant effects by decreasing protein oxidation.
Table 2. Markers of oxidative stress.
C
CKD
CKDA
CKDL
Oxidized proteins in the cortex
DNPH nmol/mg protein)
0
1
1
1
.90 ± 0.30
122.2 ± 20.99 *
59.87 ± 4.06 *,+
76.7 ± 6.29 *,+
(
Oxidized proteins in the medulla
DNPH nmol/mg protein)
.09 ± 0.30
.27 ± 0.28
.06 ± 0.36
74.42 ± 1.82 *
6.18 ± 0.32 *
6.13 ± 1.12 *
36.27 ± 5.19 *,+
2.26 ± 0.4 +
63.04 ± 3.46 *,+
2.04 ± 0.88 +
0.92 ± 0.21 +
(
Lipid peroxidation in the cortex
4HNE nmol/mg protein)
(
Lipid peroxidation in the medulla
4HNE nmol/mg protein)
1.88 ± 0.28 +
(
C: control; CKD: chronic kidney disease; CKDA: chronic kidney disease, allicin-treated; CKDL: chronic kidney
+
disease, losartan-treated. Data represent the mean
p < 0.05 versus CKDA.
±
SEM, n = 6. * p < 0.05 versus control; p < 0.05 versus CKD;
#
Additionally, we analyzed the expression of Nrf2, Keap1, catalase (CAT), superoxide dismutase
SOD), and HO-1 proteins by immunoblotting. In the cortex from the group with CKD, the expression
(
of Nrf2 was decreased compared with that of the control group. By contrast, the expression of

Int. J. Mol. Sci. 2017, 18, 1980
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Keap1 was higher in the CKD rats with respect to the control group (Figure 6). The allicin or
losartan treatments increased the expression of Nrf2, although both treatments down-regulated Keap1
expression. Nevertheless losartan showed better effectiveness than allicin in the down-regulation of
Keap1 (Figure 6). Moreover, the expression of CAT and SOD decreased in the cortexes of the CKD
animals. The expression of HO-1 was induced in the CKD group compared with the control group,
where the expression of this protein was not observed (Figure 6).
C
CKD CKDA CKDL
C
CKD CKDA CKDL
Nrf2
Keap1
β-Actin
β-Actin
+
*
1
.25
.00
.75
.50
.25
.00
1.5
1.2
0.9
0.6
0.3
0.0
*
1
#
+
*
0
*
+
0
#
0
*
*
+
0
(
a)
(b)
C
C
CKD CKDA CKDL
C
CKD CKDA CKDL
CKD CKDA CKDL
HO-1
CAT
SOD
β-Actin
β-Actin
β-Actin
0
.6
.5
.4
.3
.2
.1
.0
0.7
+
+
0.7
*
0.6
0.5
0.4
0.6
0
0.5
*
0
+
*
0.4
0.3
0.2
0.1
0.0
+
0
0.3
0.2
0.1
0
*
*
*
0
+
+
*
0
0.0
(
c)
(d)
(e)
Figure 6. Analysis of antioxidant pathway Nrf2/Keap1 in the renal cortex. (
a
) Nuclear factor
) catalase (CAT);
e) heme oxygenase-1 (HO-1). Representative immunoblot and
(erithroid-derived 2)-like 2 (Nrf2); (b) Kelch-like ECH-associated protein 1 (Keap1); (c
(
d) superoxide dismutase (SOD); (
quantitative analysis of relative protein expression. Control, C; chronic kidney disease, CKD; chronic
kidney disease, allicin-treated, CKDA; chronic kidney disease, losartan-treated, CKDL. Values are
presented as the mean
versus CKDA.
±
SEM of n = 6. * p < 0.05 versus C; + p < 0.05 versus CKD; # p < 0.05
The allicin or losartan treatments restored the down-regulation of CAT and SOD, but the
effectiveness of losartan was better than that of allicin (Figure 6). In the case of SOD expression,
allicin and losartan were equally effective in restoring the levels of SOD in the cortex (Figure 6).
The expression of HO-1 was down-regulated by the allicin or losartan treatments. However, these
effects did not reach the levels observed in the control group. We did not observe differences between
allicin and losartan in SOD and HO-1 expression levels (Figure 6).
It has been described that hypertension and oxidative stress in the kidneys is associated with an
alteration in endothelial nitric oxide synthase (eNOS) expression. To address this issue we assessed the
eNOS expression by immunoblotting. In the CKD group, the expression of eNOS was decreased in
comparison with the control group (Figure 6). However treatment with allicin or losartan increased
the eNOS expression, with allicin being more efficient than losartan (Figure 7).

Int. J. Mol. Sci. 2017, 18, 1980
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C
CKD
CKDA
CKDL
eNOS
β-Actin
0
.5
.4
.3
.2
.1
.0
*
+
0
#
*
0
+
0
0
*
0
Figure 7. Renal expression of endothelial nitric oxide synthase (eNOS) in the cortex. Representative
immunoblot and quantitative analysis of relative protein expression. Control, C; chronic kidney disease,
CKD; chronic kidney disease, allicin-treated, CKDA; chronic kidney disease, losartan-treated, CKDL.
Values are means SEM of n = 6. * p < 0.05 versus C; + p < 0.05 versus CKD; # p < 0.05 versus CKDA.
2
.7. In Silico Analysis of AT1-Losartan and -Allicin Interactions
On the basis of the similar degree of overall nephroprotection provided by losartan and allicin
treatments at the physiological, histological, functional, and molecular levels in the rat model of
CKD, it was conceivable that both compounds could act by a similar mechanism. Since losartan
is a first-choice antihypertensive drug that acts by inhibiting the activation of AT1 receptors that,
upon stimulation with angiotensinogen II, promote vasoconstriction, we carried out an in silico
analysis to assess if allicin could be interacting with AT1 in the same manner. For this, protein-ligand
docking predictions and the most favored complex energies (∆G) were obtained using Rattus norvegicus
angiotensin II receptor type 1A (RnAgtR1A) and Homo sapiens angiotensin II receptor type 1 (HsAgtR1)
as AT1 receptor homologs and losartan or allicin as ligands. As expected, the structural comparison of
human and rat AT1 counterparts showed a striking similarity of 95.6% (the threading metrics (TM)
score was 0.9560, normalized with RnAgtR1A: Figure 8A). Interestingly, the most favored docking
positions for losartan (
∆
G =
−
10.172 kCal/mol) and allicin (
∆
G =
−
6.839 kCal/mol) in RnAgtR1A
were located at the same transmembrane region, near to the extracellular domain of the receptor, where
olmesartan experimentally complexes in the reference crystal structure of human AT1 (Figure 8B,
dotted square). As a reference, olmesartan displays its most favored docking with RnAgtR1A at
∆
G = −10.158 kCal/mol.
At the sequence level, RnAgtR1A and HsAgtR1 share the seven inhibitor-interacting residues
(Tyr35, Trp84, Val108, Ser109, Arg167, Phe182, and Ile288) of which Arg167 is an interesting feature.
As can be seen in Figure 8C, the oxygen atom of allicin is able to form hydrogen bonds with H12 and
H22 of Arg167 that might contribute to a significant strengthening of the receptor-inhibitor interaction.
Remarkably, the unique oxygen atom of losartan trends directly towards H12 and H22 of Arg167.
This notion is further supported by two additional observations: (a) the optimal docking of the
oxygen-free precursor of allicin, namely diallyl disulfide (
∆
G =
−
6.316 kCal/mol), exhibits a strikingly
different conformation that is farthest from Arg167 (Figure 8D) and (b) olmesartan, a hydrolyzable
version of a sartan-type AT1 inhibitor, has three oxygen atoms with which O1 is able to form hydrogen
bonds, as allicin’s oxygen does (Figure 9).
Altogether these bioinformatics analyses suggest that the comparable nephroprotective effects of
losartan and allicin indicate a common mechanism involving AT1 receptors.

Int. J. Mol. Sci. 2017, 18, 1980
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Figure 8. In silico analyses of the interactions of AT1R homologs with losartan and allicin. (A) Protein
structure alignment of rat (RnAgtR1A, light brown) and human (HsAgtR1, green) DNA, showing
their high similarity; ( ) Docking of allicin (purple) and losartan (green), both in stick representation,
at the transmembrane region (dotted square) just beneath the extracellular domain of RnAgtR1A
blue). The intracellular domain of the receptor is shown in red; ( ) Docking of allicin (purple) and
B
(
C
losartan (green), each with the oxygen atom in red, with the putative inhibitor-interacting aminoacids
of RnAgtR1A. Note the hydrogen bonds (green lines) formed between oxygen from allicin and H12
and H22 of Arg167; (D) Docking of allicin (purple) and its oxygen-free precursor diallyl disulfide (blue)
at the niche of putative inhibitor-interacting amino acids, where the bridge-forming oxygen atom of
allicin drives a distinct conformation and accommodation of this ligand.
Figure 9. Optimal docking of allicin (purple), losartan (green), and olmesartan (blue), all in stick
representation, in RnAgtR1A in which hydrogen bonds are formed by the oxygen atoms O1 of
olmesartan (green lines) and of allicin (cyano lines) with hydrogens H12 and H22 from arginine
1
67 (orange), whilst the unique oxygen atom of losartan is docked more distantly.

Int. J. Mol. Sci. 2017, 18, 1980
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3
. Discussion
Hypertension is a hemodynamic characteristic of CKD and is considered one of the most important
factors in determining the rate of deterioration of renal function by worsening glomerular injury
and proteinuria [ ]. It is believed that hypertension and CKD may be mutually both the cause
and consequence of each other [ ]. Recently, we reported that allicin treatment improved cardiac
7
1
dysfunction and decreased hypertension, vascular reactivity to Ang II, and the oxidative stress in a
way dependent on the Nrf2/Keap1 system [18]. This previous study showed the therapeutic potential
of allicin for a physiopathological condition in which the cardiac and/or renal functions are involved
and compromised. However, we did not delve into the study of the effects of allicin on the kidney.
Thus, the aim of the present study was to contribute to the knowledge of this issue and, on the other
hand, to compare the therapeutic efficacy of allicin with losartan, a well-known and common drug
used in the clinic to treat hypertension.
High BP is a hemodynamic characteristic of CKD, which could accelerate the progression of
renal dysfunction by worsening glomerular injury and proteinuria which, in turn, promotes further
glomerular and tubulointerstitial injury. As a consequence, a fall in the glomerular filtration rate may
ensue [
patients and experimental models, and both are frequently used as an index of renal damage,
specifically as glomerular and tubular markers, respectively [19 20]. In line with these reports, we
7]. Furthermore, albuminuria and urinary excretion of NAG are increased in hypertensive
,
observed an increase in systolic blood pressure at the third week of follow-up, which was aggravated
at the sixth week after renal ablation, and this was associated with a worsening of glomerular and
tubular injury (an increase in albuminuria and urinary NAG).
The main mechanism through which albuminuria occurs in CKD involves the alteration of the
structure and function of the glomerular filtration barrier. Nephrin is an important transmembrane
protein, fundamental for the podocyte slit diaphragm function due to its regulation of renal filtration
by selectively allowing small molecules like ions to pass through, while excluding the passage of large
molecules like proteins [21
group. Other studies in hypertensive patients and experimental models have described increased
nephrin expression in early stages [23 26]. In fact, in patients with CKD, a loss of nephrin occurs
,22]. Our results showed an increase in nephrin expression in the CKD
–
despite the increased level of nephrin mRNA [23]. In contrast, the longer-term studies report that,
in the advanced stages of the disease, nephrin expression is reduced [23–26].
It has been suggested that the up-regulation of nephrin may be compensatory to protect against
podocyte injury. However, in the long-term, the mechanism may not be able to counteract the injury,
and the downregulation may be a result of podocyte injury, which progressively aggravates renal
function and proteinuria.
We hypothesized that hemodynamic forces induced the loss of nephrin in urine, allowing the
passage of albumina and other small molecules. To compensate this loss and to maintain the integrity
of the glomerular filtration barrier, the kidneys increased the nephrin expression. Our results suggested
structural alterations; therefore, we studied the renal histology. The kidney injury was clearly shown
by the histologic analysis at the glomerular and tubular levels, which was attenuated by the allicin or
losartan treatments at both the biochemical and structural levels.
Allicin and losartan showed the same effectiveness to decrease hypertension, albuminuria, and
urinary NAG excretion. The level of blood pressure reduction achieved with allicin was comparable
to that of common antihypertensive medication (
respectively) with respect to untreated CKD, which showed
results indicated that allicin had a significant effect in reducing blood pressure, which is consistent
with previous studies, even in hypertensive patients [15–17].
−
15 and
−
30.2 mmHg over three and six weeks,
180 mmHg at the end of the study. The
≥
In addition, the expression of nephrin was decreased with allicin and losartan. We think that, due
to the reduction in hemodynamic pressure on the glomerular filtration barrier, this remained stable
and the synthesis of nephrin decreased, finally contributing to the improvement of renal function.

Int. J. Mol. Sci. 2017, 18, 1980
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The RAS plays a pivotal role in many of the pathophysiological changes that lead to the
progression of renal disease. These changes are mediated by the interaction of AngII with its receptors.
In this study, we find decreased AT1 and AT2 receptor expression in the kidneys. Other studies
have reported a decrease in AT1R and AT2R expression in renal hypertension [20,27–29]. It has
been hypothesized that the AT1R expression decreased as a protective mechanism to counteract
the deleterious effects of hyperactive intrarenal RAS. Moreover, it is well documented that AT1R in
blood vessels and the kidneys are under negative feedback modulation by Ang II. High systemic
and renal Ang II levels downregulate, whereas the low concentration of plasma and renal Ang II
upregulates AT1R in the kidneys [29
Ang II stimulation is particularly important in disease states characterized by an activated RAS [29
–
31]. This downregulation of AT1R expression observed with
30].
,
Allicin and losartan treatment prevents the down-regulation of the CKD induced angiotensin II
receptors. To our knowledge, this is the first study in which the effect of allicin on angiotensin
receptors in the kidneys is described. Despite the CKD group having reduced AT1 receptor expression,
this model is still very sensitive to the renoprotective effects of AT1 receptor antagonists. Moreover,
strong support for the likely interaction of allicin with AT1R was provided by in silico modelling and
docking analyses in which this garlic derivative optimally complexes at the same site where typical
AT1R inhibitors (losartan, olmesartan) promote hypertension blocking and where an oxygen atom
from these compounds could form hydrogen bonds (especially with Arg167), thereby strengthening
the receptor-inhibitor interactions and pharmacological effects. This last notion would be important
in designing drugs to interact with AT1R, as exemplified by olmesartan, an ester prodrug that, upon
hydrolysis, displays several oxygen atoms able to form hydrogen bonds with Arg167 in AT1R. However
another potential mechanism for allicin nephroprotection might include its free radical-scavenging
properties [32], even in CKD, as described below, as well as an inhibitory effect on angiotensinogen
converting enzyme (ACE) activity, which has already been reported for garlic extracts in normal and
diabetic rats [33].
On the other hand, oxidative stress in CKD and hypertension commonly accompanies both
disorders. Our results showed increased lipid and protein oxidation, which are indicators of oxidative
stress. We evaluate the expression of antioxidant enzymes as an indicator of balance between the
generation and the elimination of free radicals and the expression of Nrf2-Keap1 a master regulator
of antioxidant genes. CAT and SOD were decreased. These results were associated with decreased
Nrf2, and its repressor, Keap1, increased. In contrast, HO-1 was increased. This can be explained
because HO-1 is a protein upregulated by several stimuli such as oxidative stress, nitric oxide, growth
factors, cytokines, modified lipids, heat shock, ischemia-reperfusion, and its substrate, heme [34
,35].
In addition, the HO-1 promoter contains binding sites for nuclear factor-kappa B (NF B), transcription
κ
factor activator protein-1 (AP-1), cAMP response element binding protein (CREB), antioxidant
response element (ARE), and Nrf2 binding. On the other hand, several signaling pathways that
include protein kinase C (PKC) or protein kinase A (PKA) and extracellular signal regulated kinase
(ERK)/mitogen-activated protein kinase (MAPK) may also regulate HO-1 at the translational level
rather than at the transcriptional level [34,35].
Both the allicin and the losartan treatments were effective in reducing alterations in CKD-induced
oxidative status in a dependent pathway of Nrf2/Keap1.
It has been reported that allicin can induce the Nrf2/Keap1 system [18,36], which could stimulate
the expression of antioxidant enzymes and decrease oxidative stress. However, to our knowledge, this
is the first study that reports the effect of allicin on the Nrf2/Keap1 pathway in the kidneys, especially
in the context of an emerging disease such as CKD.
Additionally, we assessed the eNOS expression as an indicator of endothelial function. Our result
showed decreased eNOS in the kidney, thus suggesting an alteration in vasodilation, which could
contribute to increased blood pressure. These findings are in agreement with those reported in other
studies [37–39]. The allicin or the losartan treatment increased the eNOS expression, but allicin
was more effective than losartan. In CKD, the excess of ROS may directly stimulate vascular

Int. J. Mol. Sci. 2017, 18, 1980
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contraction or reduce nitric oxide, contributing to hypertension [40]. Moreover, there is an increase of
asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase [41]. These two factors are
responsible for a decrease in nitric oxide activity in CKD, which contribute to endothelial dysfunction,
vasoconstriction, and, therefore, increased blood pressure.
Several reports indicate that allicin byself shows antioxidant properties by scavenging free
radicals [42] and hydroxyl radicals (OH ) [43]. Interestingly, Liu et al. [44] described that allicin
•
reduces AngII-induced oxidative stress in cardiac hypertrophy in both in vitro and in vivo models.
Other studies have demonstrated that allicin prevents ROS damage by upregulating the phase II
detoxifying enzymes and increasing the cellular glutathione levels [45].
Given the well-documented role of ROS in hypertension, it is reasonable to propose that allicin
ameliorates hypertension by inhibiting cellular oxidative stress.
On the other hand, it has been suggested that allicin acts as an antihypertensive mainly due
to decomposing rapidly to its degradation products. On the other hand, the vasoactivity of garlic
compounds is synchronous with hydrogen sulfide (H S) production, and their potency to mediate
2
relaxation increases with (H S) yield, a potent gaseous signaling molecule with the capacity to control
2
blood pressure by the relaxation of smooth muscle cells [46]. In anesthetized casts with intact chests
and the isolated lungs of rat under constant flow conditions, allicin showed vasodilatory activity at the
systemic and pulmonary levels [47]. Allicin rapidly decomposes, mainly to diallyl sulfide (DAS), diallyl
disulfide (DADS), diallyl trisulfide (DATS), and ajoene [13]. However, after consumption, neither
allicin nor its metabolites have been found in blood or urine [48], suggesting that the metabolites of
allicin are rapidly metabolized.
Altogether, the evidence from the present study suggests that the nephroprotective effects of
allicin may be partly due to antihypertensive and antioxidant effects, which preserve the function and
renal structures in hypertensive rats with CKD induced by 5/6 Nx.
4
. Methods
4
.1. Reagents
Diphenyl dichloromethane, diallyl disulphide, hydrogen peroxide, 4-nitrophenol, methanesulfonic
acid, tetramethoxypropane, dinitrophenylhydrazine, 1-methyl-2-phenylindole 4-nitrophenyl-N-acetyl-
-D-glucosaminide, acetonitrile, methanol, and guanidine were purchased from Sigma (St. Louis,
MO, USA). The anti-angiotensin II type 1 receptor antibody (ab124734) and anti-angiotensin II type 2
receptor antibody (ab92445) were purchased from Abcam (Cambridge, MA, USA). The -actin antibody
β
β
GTX109639, eNOS antibody GTX54637, KEAP1 antibody GTX60660, and NRF2 antibody GTX103322
were purchased from GeneTex Inc. (Irvine, CA, USA). The nephrin antibody ALX-810-016-R100 was
purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The catalase antibody sc-34281,
heme oxygenase 1 antibody sc-1797, and superoxide dismutase antibodysc-8637 were purchased from
Santa Cruz Biotechnology Inc. (Dallas, TX, USA). All other chemicals used herein were of the highest
analytical grade available.
4
.2. Experimental Design
Male Wistar rats weighing 280 to 300 g were used. The rats were divided into four groups (n = 12
each): sham-operated animals served as a control (C), chronic kidney disease (CKD) (induced by renal
ablation (5/6 nephrectomy)), CKD+allicin (40 mg/kg/day pathway oral) (CKDA), and CKD+Losartan
(20 mg/kg/day pathway oral) (CKDL). All experimental groups were fed with commercial rodent
pellets (PMI Nutrition International, Inc., LabDiet 5008, Richmond, IN, USA) and water ad libitum for
six weeks.

Int. J. Mol. Sci. 2017, 18, 1980
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4
.3. Ethics Statement
This study was performed in accordance with the Guide for the Care and Use of Laboratory
Animals, published by the U.S. National Institute of Health and approved by the Research Committee
of the National Institute of Cardiology Ignacio Chávez and by the Mexican Federal Regulation for
animal experimentation and care (NOM-062-ZOO-2001) and for the disposal of biological residues
(NOM-087-ECOL-1995).
4
.4. Allicin
Allicin (diallyl thiosulfinate) was produced by the oxidation of diallyl disulphide, as previously
reported [32]. In brief, 1 g diallyl disulphide was dissolved in 5 mL acetic acid under stirring in an
ice bath. Hydrogen peroxide (1.5 mL, 30% v/v) was added stepwise, and the reaction was allowed to
◦
proceed for 30 min. Afterwards, the reaction was kept at 13 C for 20 min; then it was put in an ice bath
again for 5 h, stopped with 15 mL distilled water at pH 6.5, and extracted with 30 mL dichloromethane.
After five extractions with 5% (w/v) Na CO (20 mL each) and three extractions with distilled water
2
3
(
20 mL each), the solvent was left to evaporate until a yellowish oil (allicin) remained. For stabilization
◦
and storage, allicin was resuspended in water at 2.5% (w/v) and kept at –70 C until used.
4
.5. Induction of Experimental Model of Chronic Kidney Disease
Under deep anesthesia with isofluorane, subtotal renal ablation was performed by the removal
of the right kidney and the selective infarction of approximately two thirds of the left kidney by
ligation of two or three branches of the renal artery (5/6 Nx). The sham operation consisted of
ventral laparotomy and the manipulation of the kidneys and renal pedicle without the destruction of
renal tissue. The muscle and skin incisions were sutured with polypropylene suture. After surgery,
the animals were returned to the animal facility for recovery [18].
4
.6. Systolic Blood Pressure (SBP)
SBP was measured in conscious restrained rats by tail-cuff plethysmography (XBP-1000 Kent
Scientific, Torrington, CT, USA). The rats were preconditioned twice before SBP was measured at the
basal period; the mean of five consecutive readings was recorded as the blood pressure, three and six
weeks after renal ablation.
4
.7. Renal Function
Three and six weeks after renal ablation, the rats were placed in metabolic cages (Nalgene,
Rochester, NY, USA) to collect 24-h urine. Urine samples were centrifuged at 5000
×
g for 15 min to
remove debris, and the supernatant was analyzed. The variables measured were creatinine and blood
urea nitrogen (BUN) (IL 300 plus, Clinical Chemistry Analyzer, Instrumentation Laboratory, Bedford,
MA, USA), microalbuminuria (Albumin Rat ELISA Kit, Abcam Cambridge, MA, USA), and urinary
N-acetyl-β-D-glucosaminidase (NAG) activity (spectrophotometric assay).
4
.8. Measurement of NAG Activity
For the determination of NAG activity in urine samples, 4-nitrophenyl-N-acetyl-β-D-glucosaminide
was used as the substrate. One unit of enzymatic activity (U) represents the amount of enzyme that
◦
hydrolyses one mol of substrate per min at 37 C. The results were expressed as units/day (U/24 h).
To evaluate the tubular injury, a set of animals was sacrificed after three weeks of follow-up after
renal ablation (six rats per group). The remaining rats were studied after six weeks of follow-up (six
rats per group). All experimental groups were maintained with a laboratory diet and water ad libitum.
The rats were euthanized by anesthesia with isoflurane and exsanguination. A blood sample
was collected and centrifuged. The plasma was frozen until used in further analyses. The kidney was
excised, washed with cold phosphate-buffered saline, and longitudinally divided into two sections:

Int. J. Mol. Sci. 2017, 18, 1980
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one part for protein work, which was excised and divided into cortex and medulla, frozen in liquid
nitrogen, and stored until further processing, and the other for hematoxylin and eosin (H and E)
staining, which was fixed in 10% formalin in phosphate-buffered saline (PBS) and embedded in
paraffin for histological studies.
4
.9. Histopathological Study
From the paraffin blocks, 4 µm sections were obtained, hematoxylin and eosin stained (HE),
and observed with a Zeiss Axiophot2 light microscope. All slides were analyzed in a blinded fashion.
For a fibrosis assessment, 10 cortex field images (200 magnification) were recorded at random
×
from Sirius Red-stained sections of each kidney. The evaluation of fibrosis focused on red-stained
areas (collagen I and collagen III) located between tubules and glomeruli, and its quantification was
performed with a computerized image analysis software (Axiovision Rel. 4.8.2, Zeiss, Jena, Germany).
Microscopic images were obtained using a digital camera mounted on a light microscope (Axiophot 2,
Zeiss, Jena, Germany). All slides were analyzed in a blinded fashion.
4
.10. Evaluation of Renal Oxidative Stress
4
.10.1. Determination of Lipid Peroxidation
4-Hydroxynonenal (4-HNE) was measured using a standard curve of tetramethoxypropane.
A solution of 1-methyl-2-phenylindole in acetonitrile:methanol (3:1) was added to heart homogenates,
and the reaction was started with 37% HCl or methanesulfonic acid plus FeCl to measure 4-HNE.
3
◦
The optical density was measured at 586 nm after 1 h of incubation at 45 C. The data were expressed
as nmol of 4-HNE per milligram of protein (nmol HNE/mg protein).
4
.10.2. Measurement of Oxidized Proteins
The presence of carbonyl groups in the proteins was measured using the reaction with
,4-dinitrophenylhydrazine (DNPH). The protein carbonyl groups were estimated by using the molar
2
−1 −1
absorption coefficient of 22,000 M
·
cm for DNPH derivatives, and their concentrations were
expressed as nmol carbonyl groups/mg protein. Guanidine solution was used as a blank.
4
.10.3. Expression of Nephrin, Nrf2/Keap1 Pathway, Endothelial Nitric Oxide Synthase (eNOS), and
Angiotensin II Receptors in Renal Cortex
The cortex was washed thoroughly with ice sold saline 10% (w/v) and later homogenized in a
Potter Elvehjem homogenizer in ice-cold 50 mM phosphate buffer at pH 7.4 containing mammalian
protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific, Waltham, MA,
USA). The homogenates were used for the determination of total protein concentration by the Bradford
method using bovine serum albumin as the standard. Equal protein concentrations (15
µ
g) were
◦
denatured in gel loading buffer at 85 C for 5 min and then loaded onto 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes
◦
and incubated at 4 C overnight with the primary antibody diluted in phosphate buffered saline
®
with the detergent Tween 20. Housekeeping protein
β-actin was used as a loading control. Positive
immunoreactive bands were quantified densitometrically and expressed as the ratio of problem to
-actin in arbitrary units. The protein bands were visualized with enhanced chemiluminescence
β
reagents (ECL Plus Western Blotting Detection System, Amersham Pharmacia Biotech, Piscataway, NJ,
USA) and analyzed, and their intensity was quantified using a Kodak Electrophoresis Documentation
and Analysis System 290 (EDAS 290).
4
.11. Bioinformatics Analyses
In order to predict the ability of typical angiotensin II receptor inhibitors (losartan, olmesartan)
and garlic compounds (allicin and diallyl diulfide) to interact with, and thereby inhibit, the function of

Int. J. Mol. Sci. 2017, 18, 1980
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rat (RnAgtR1A) and human (HsAgtR1) counterparts of the angiotensin II receptor, in silico approaches
of molecular docking were performed using the Swiss Dock [49] web service. This tool first calculates,
orders in clusters, and delivers a set of 256 viable positions between one rigid target (protein) and
one flexible ligand (compound), which are input in Protein Data Bank (PDB) and Tripos molecule
structure (MOL2) formats, respectively. Of these predictions, the most favored docking was selected
by considering the lowest (i.e., most negative value) Gibbs free energy (∆G). In these analyses, the
protein structures of RnAgtR1A and HsAgtR1 were raised using the Swiss Model server [50], whilst
the crystal structure of human angiotensin receptor II type 1 (PDB ID: 4YAY, ligand: olmesartan) was
used as the structure of reference to identify inhibitor-interacting amino acids. All predictions obtained
from SwissDock were visualized and edited using the University of California at San Francisco
(UCSF)-Chimera package v. 1.10.1 (UCSF, San Francisco, CA, USA). Structural comparisons of the
modelled proteins were carried out using the TM-Align tool accessible via the I-TASSER server [51].
4
.12. Statistical Analyses
All the values are expressed as mean
was determined by one-way ANOVA, followed by Bonferroni analysis using Prism 5 (GraphPad,
±
standard error of the mean (SEM). Statistical significance
San Diego, CA, USA) software. Differences were considered significant when p < 0.05.
5
. Conclusions
Allicin showed antihypertensive, antioxidant, and nephroprotective effects. The beneficial effects
showed by allicin are similar to or better than those exerted by losartan. In fact, the effect of allicin
on blood pressure and renal function is comparable to reductions seen with losartan, a prescription
drug commonly used as a first-line therapy. Therefore, allicin may be a useful tool for the treatment of
emerging diseases such as CKD.
Acknowledgments: This study was supported by Fondos del Gasto Directo Autorizado a la Subdirección de
Investigación Básica, Instituto Nacional de Cardiología “Ignacio Chávez”.
Author Contributions: Ehécatl Miguel Ángel García Trejo, Abraham Said Arellano Buendía, Omegar Sánchez Reyes
and Fernando Enrique García Arroyo performed the animal treatments, protein isolation, Western blot, and lipid
and protein oxidation. Raúl Arguello García performed the allicin and bioinformatics analyses, the analysis of
results, and the review of the manuscript. María Lilia Loredo Mendoza performed the histomorphometrical
analysis and contributed to the analysis of the results and to the review of the manuscript. Edilia Tapia
performed the surgical procedure to establish the CKD model, the analysis of the results, and the review of
the manuscript. Laura Gabriela Sánchez Lozada contributed to the analysis of the results and the critical review of
the manuscript. Horacio Osorio Alonso conducted the study design, statistical analysis, data interpretation, and
manuscript preparation. All authors contributed to the draft of the manuscript and agreed on the final version of
the manuscript.
,
Conflicts of Interest: The authors declare no conflicts of interest.
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