Journal of Functional Foods 43 (2018) 37–43

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Journal of Functional Foods

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Arthrospira maxima (Spirulina) and C-phycocyanin prevent the progression T

of chronic kidney disease and its cardiovascular complications
Ivonne Nayelli Memije-Lazaroa,b, Vanessa Blas-Valdiviaa, Margarita Franco-Colínb,
⁎
Edgar Cano-Europab,
a
Laboratorio de Neurobiología, Departamento de Fisiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Departamento de Fisiología, Escuela
Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Av. Wilfrido Massieu s/n, Esq. Manuel L. Stampa, Colonia Unidad Profesional Adolfo López Mateos,
Delegación Gustavo A. Madero, Código Postal 07738 Ciudad de México, Mexico
b
Laboratorio de Metabolismo, Departamento de Fisiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Departamento de Fisiología, Escuela
Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Av. Wilfrido Massieu s/n, Esq. Manuel L. Stampa, Colonia Unidad Profesional Adolfo López Mateos,
Delegación Gustavo A. Madero, Código Postal 07738 Ciudad de México, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: Chronic kidney disease (CKD) is a worldwide public health problem. CKD progression causes cardiovascular
Arthrospira maxima complications that involve substantial costs in terms of medical managing and that end with the patient's death.
Spirulina Consequently, it is necessary to develop new therapeutic strategies to reduces the CKD-related causes of car-
Phycocyanin diovascular complications. Nutraceuticals such as Arthrospira maxima (Spirulina) and C-phycocyanin have been
Chronic kidney disease
used in the treatment of renal and cardiovascular disease. Thus, this study aimed to evaluate wheter the ad-
Hypertension
ministration of A. maxima or C-phycocyanin prevents the progression of CKD and cardiovascular complications
in rat CKD model. We observed that A. maxima and C-phycocyanin reduced CKD-related causes of hypertension,
left ventricular hypertrophy, renal dysfunction and oxidative stress in the kidney and heart. In conclusion, A.
maxima or C-phycocyanin could be used to delay CKD because they prevent the progression of CKD and the
associated cardiovascular complications.

1. Introduction
Chronic kidney disease (CKD) is defined as an abnormality of the
kidney’s structure or function that is present for at least three months
with implications for health, and it is the result of various chronic de-
generative diseases such as diabetes mellitus and hypertension (Inker
et al., 2014). This disease is a worldwide public health problem. It has
been reported that CKD is one of the leading causes of morbidity,
mortality, and high medical costs among adults in the United States. In
2010, the cost of end-stage CKD was about $32.9 billion, and the cost of
first-stage CKD was about $48 billion (Hoerger et al., 2015). In Mexico,
the reported incidence of CKD is about 37% with a rate of 1142 cases
per million people. Also, 52,000 patients receive substitution therapies
such as peritoneal dialysis or hemodialysis (Méndez-Durán et al., 2014).
There is a strong relationship between the progression of CKD and
cardiovascular events (CV). In fact, cardiovascular diseases are the
leading cause of 50% of deaths in patients with CKD due to numerous
structural and functional cardiovascular disturbances such as en-
dothelial dysfunction, arterial stiffness, left ventricular hypertrophy
(LVH), and vessel wall remodeling. All these physiopathological events
occur from the early stages of CKD. Also, CKD causes systemic hy-
pertension as a compensatory mechanism to prevent the reduction of
the glomerular filtration rate caused by the loss of glomeruli. However,
this is a positive feedback loop that participates in the progressive in-
crease of blood pressure and renal damage (Alani, Tamimi, & Tamimi,
2014). Moreover, one natural event present during the progression of
CKD is oxidative stress. Oxidative stress is defined as a disturbance
existing in the oxidant system and the antioxidants system inside the
cells. The oxidant system components are the reactive species derived
from oxygen (ROS), nitrogen (RNS), and some metals that exist in the
cells. The ROS has high reactivity, and it includes free radicals such as
superoxide anion (O2%−) and hydroxyl anion (OH%−), as well as
non‐radicals such as hydrogen peroxide (H2O2). Concerning the anti-
oxidant system, there are two subsystems. The non-enzymatic system
contains vitamin A, C, and E; reduced glutathione; α-lipoic acid, among
other components. Meanwhile, the antioxidant enzymatic system con-
tains catalase, superoxide dismutase, glutathione peroxidase (GPX), and
peroxidase (GR), among other components (Lushchak, 2011). In the
early CKD stage, renal damage and mitochondrial dysfunction generate
a vicious functional circle responsible for the overproduction of ROS,

⁎
Corresponding author.
E-mail addresses: edgarcanoeuropa@yahoo.com.mx, ecanoe@ipn.mx (E. Cano-Europa).

https://doi.org/10.1016/j.jff.2018.01.013
Received 18 July 2017; Received in revised form 4 January 2018; Accepted 14 January 2018
1756-4646/ © 2018 Published by Elsevier Ltd.
I.N. Memije-Lazaro et al.
which disturbs the mitochondrial calcium homeostasis. These events
have been associated with podocyte injury leading to proteinuria and
the transition of the tubular epithelial cells to mesenchymal (Signorini,
Granata, Lupo, & Zaza, 2017). Also, CKD causes an overactivation of the
renin-angiotensin system that increases the production of ROS in the
endothelium and cardiomyocytes due to the overactivity and over-
expression of the NADPH oxidase and xanthine oxidase (G.-X. Zhang,
Lu, Kimura, & Nishiyama, 2007). Also, in the kidney and cardiovascular
systems of rats with CKD, a reduction has been observed in the activity
and expression of the antioxidant enzymes such as catalase and GPX.
Thus, the CKD-related causes of oxidative stress in the kidney and the
cardiovascular system promote carbohydrates, proteins, nucleic acids,
and lipids oxidation while also activating signaling pathways associated
with MAPK, p38, and c-jun, among other factors, which are related to
cardiovascular complications such as left ventricular hypertrophy
(Sindhu et al., 2005). Therefore, one therapeutic strategy to delay CKD
is the use of antioxidants to prevent CKD-related causes of renal and
cardiovascular system oxidative stress, which is associated with the
progression of CKD and its cardiovascular complications (Vaziri, 2004).
With all the antecedents mentioned above, the early and timely iden-
tification and treatment of CKD are necessary to prevent disease pro-
gression and reduce the risk of cardiovascular morbidity and mortality.
The use of antioxidants is an important therapeutic strategy that could
be extensively studied for use in the future for the benefit of patients. In
this way, some research groups have proposed the use of nutraceuticals
to delay CKD. Arthrospira maxima (Spirulina) and C-phycocyanin are
considered nutraceuticals. They have been used in the treatment of
acute renal damage (Fernandez-Rojas et al., 2014; Rodriguez-Sánchez,
Ortiz-Butrón, Blas-Valdivia, Hernández-García, & Cano-Europa, 2012)
and cardiovascular disorders (Khan et al., 2006; Wu et al., 2016) be-
cause of their antioxidant activity. Spirulina and C-phycocyanin are
potent antioxidants because they are free radical scavengers. It has been
demonstrated that Spirulina and C-phycocyanin scavenge ROS and RNS
through in vitro assays (Romay et al., 1998), and they also prevent the
increase of oxidative stress markers associated with drug-related causes
of cellular damage (Bayomy, Abdelaziz, Said, Badawi, & El-Bakary,
2016; Rodriguez-Sánchez et al., 2012; Sinanoglu, Yener, Ekici, Midi, &
Aksungar, 2012).
Arthrospira maxima (Spirulina) is a unicellular filamentous blue-
green cyanobacterium. It has been used as food by humans and other
animals because of its high nutritional value. It contains proteins, es-
sential amino acids, minerals, and essential fatty acids ω-3 and ω-6,
among other components. It also contains vitamins A, C, and E, and
accessory pigments such as phycobiliproteins. One of the most abun-
dant phycobiliproteins in A. maxima is the C-phycocyanin, which con-
tains an open-chain tetrapyrrole chromophore known as phycocyano-
bilin (Kumar, Dhar, Pabbi, Kumar, & Walia, 2014). Therefore, both
Spirulina and C-phycocyanin have great therapeutic potential and
should be studied as a CKD treatment. Thus, this study aims to de-
termine whether the administration of A. maxima (Spirulina) or C-
phycocyanin prevents the progression of CKD and its cardiovascular
complications using a heuristic model of CKD (nephrectomy 5/6, NFx
5/6). The NFx 5/6 is a biological model that involves employing a
surgical reduction of 5/6 of the total renal mass, which replicates the
CKD-related causes of renal dysfunction and cardiovascular complica-
tions.
2. Material and methods
2.1. Animals
Thirty-six male Wistar rats were used. Animals weighed between
250 and 300 g. They were housed in groups of six in a Plexiglas cage,
with food and water ad libitum, in a room with constant temperature
(21 ± 2 °C) and a 12 h light:12 h dark cycle (lights on at 0800).
All experimental procedures described in this study followed the
38
Journal of Functional Foods 43 (2018) 37–43
guidelines of the Laws and Codes of Mexico in The Seventh Title of the
Regulations of the General Law of Health Regarding Health Research,
and the Mexican Official Standard NOM-062-ZOO-2001 of the technical
specifications for the production, care, and use of laboratory animals.
2.2. A. maxima (Spirulina) used during treatment
The Arthrospira maxima used during the treatment were donated by
Alimentos Esenciales para la Humanidad S. A de C. V. (Mexico).
2.3. Cultivation of A. maxima for obtaining of C-phycocyanin
The growing of the cyanobacterium Arthrospira maxima (Spirulina)
to obtain C-phycocyanin was done as previously described but with
some modifications (Rodriguez-Sánchez et al., 2012). Briefly, the cya-
nobacterium was grown in repeated batches in Zarrouk medium, which
contains 150 mM NaHCO3, 2.87 mM K2HPO4, 29.4 mM NaNO3, 5.7 mM
K2SO4, 17.1 mM NaCl, 1.4 mM MgSO4, 0.4 mM CaCl2, 36.9 M FeSO4,
0.2 mM EDTA, 46.2 M H3BO3, 9.3 M MnCl2, 0.95 M ZnSO4, 2.03 M
Na2MoO4, 0.49 M Ca(NO3)2, and 0.77 M CuSO4 at 35 ± 2 °C. The
culture was supplied with the constant illumination of 180 μE/m2 s
provided by a halogen lamp over the course of 24 h. The aeration was
constant using an air pump.
One part of the cell mass was dried and was used for the treatment.
Meanwhile, another part was used to obtained phycocyanin. Briefly, 5 g
of the washed cell mass was re-suspended in 20 ml of distilled water
and was frozen at -20 °C over the course of 24 h twice. The resultant
slurry was centrifuged between seven and ten centrifugation cycles at
2500g for 10 min to remove the cell debris. The phycobiliproteins in the
supernatant were spectrophotometrically characterized as described by
Bennett and Bogorad (1973) using the following equations:
Phycocyanin ( )=
mg
ml
A 620 − 0.474A 652
5.34
Allophycocyanin ( ) =
mg A 652 − 0.208A 620
ml 5.09
Phycoerythrin ( ) =
mg A 652 − 0.241 [phycocyanin] − 0.89 [allophycocyanin]
ml 9.62
Twenty-five mL of the phycobiliproteins-rich extract was put on a
column (33-cm length × 4.7-cm diameter) of Sephadex G-250 (Sigma,
Co.) that had been equilibrated with 100 mM PB pH 7.4. The Sephadex
column was eluted with a PB pH 7.4 with a linear gradient from
100 mM to 6.5 mM. Five fractions were obtained and were precipitated
with a saturated solution of (NH4)2SO4 at 4 °C with agitation. After that,
each fraction was kept in the dark for 48 h at 4 °C. Subsequently, the
fractions were dialyzed in distilled water at 4 °C for 48 h in the dark,
were then dehydrated, and were then collected in tubes and stored at
−70 °C.
The dialyzed fractions four and five were put on a DEAE–cellulose
(Sigma, Co.) column (3.3-cm length × 4.7-cm diameter) that had been
equilibrated with 50 mM acetate buffer pH 5.5. The column was eluted
with a 50 mM acetate buffer pH 5.5. The bluish fractions were col-
lected, and they were precipitated with added, finely powdered
(NH4)2SO4 to achieve a saturated solution at 4 °C for 48 h in the dark.
They were then dialyzed in distilled water at 4 °C for 48 h in the dark
and were dehydrated and stored at -70 °C until characterization.
2.4. Rat model of chronic renal failure
The animals were randomly assigned to six groups: (1) sham; (2)
sham + Arthrospira maxima (Spirulina; 1 g/kg/d, i.g.); (3) sham + C-
phycocyanin (100 mg/kg/d, i.g.); (4) nephrectomy 5/6 (NFX); (5)
NFX + A. maxima; and (6) NFX + C-phycocyanin (100 mg/kg/d, i.g.).
Nephrectomy was done on rats anesthetized with sodium pento-
barbital-xylazine (16 and 20 mg/kg, respectively). Also, to prevent the
side effects of anesthesia, they were given atropine-caffeine (2 and
20 mg/kg, respectively). Aseptically, a ventral laparotomy was
I.N. Memije-Lazaro et al. Journal of Functional Foods 43 (2018) 37–43

performed, and two of the three branches of the left kidney artery were Table 1
occluded so that approximately 2/3 of the left kidney was infarcted; Effect of A. maxima and C-phycocyanin administration on cardiovascular alterations in 5/
6 nephrectomized (5/6 NFx) rats.
later, the right kidney was removed. After surgery, mefenamic acid
(10 mg/kg, i.g.) and enrofloxacin (4 mg/kg, i.m.) were administered Groups Systolic blood Interventricular Left ventricular
over two days to alleviate pain and prevent infection. pressure Septal Thickness area
After five weeks of treatment, the mean arterial pressure and renal (mmHg) (mm) (mm2)
function were evaluated. The animals were sacrificed, and their kidneys
Sham 113 ± 6.3 2.8 ± 0.1 5.6 ± 0.5
and hearts were dissected. The organs were frozen at −70 °C until they 5/6 NFx 207 ± 4.6* 4.2 ± 0.1* 2.3 ± 0.55*
were used to measure oxidative and redox environment markers. Sham + A. 118 ± 7.2 2.8 ± 0.1 4.6 ± 0.7
maxima
2.5. Seric and urinary chemistry 5/6 NFx + A. 156 ± 6.9*,** 3.6 ± 0.2*,** 3.9 ± 0.3*,**
maxima
Sham + C- 120 ± 0.8 2.6 ± 0.2 5.4 ± 0.5
After five weeks of treatment, each rat was individually placed in phycocyanin
metabolic cages for collection of 24-h urine. On this day, blood samples 5/6 NFx + C- 130 ± 7.0 *,**
2.5 ± 0.2 4.9 ± 0.5
were taken from the caudal vein. Protein concentration in urine, serum phycocyanin
uric acid, plasma creatinine, and creatinine clearance were measured
The data represent the mean of six independent data ± SE. One-way ANOVA and
using Randox kits.
Student-Newman-Keuls post hoc.
* P < .05 vs sham.
2.6. Cardiovascular evaluations ** P < .05 vs 5/6 NFx.

The blood systolic, diastolic, and mean pressure in conscious rats
were determined by a noninvasive method with a digital plethysmo-
graph tail (Digital Pressure Meter Le 5002 LETICA®, Panlab, S.L.
Barcelona, Spain). The extent of left ventricular hypertrophy was also
determined. Briefly, the hearts were cut into transverse slices two
millimeters in length to measure the thickness of the interventricular
septum and the left ventricular area using the program Image J.
2.7. Oxidative and REDOX environment markers
The organs were homogenized in 3 ml of 10 mM phosphate buffer
(pH 7.4), which was used for all biochemical tests as previously de-
scribed (Rodriguez-Sánchez et al., 2012).
The lipid peroxidation (LP) was evaluated by the formation of a
lipid-soluble fluorescence. Briefly, 500 µl aliquots were added to 4 ml of
chloroform-methanol (2:1, v/v). They were then agitated to allow for
the separation of the aqueous and chloroform phases. The samples were
kept at 4° C for 30 min and protected from light, after which time the
aqueous phase was aspirated and discarded. Finally, 2 ml of the organic
phase (chloroform) were taken, and the fluorescence was determined
using an RF5000U Shimadzu Spectrophotometer at 370 nm (excitation)
and 430 nm (emission) wavelengths. The results were expressed as re-
lative fluorescence units (RFU) per milligram of protein, as previously
described (Cano-Europa et al., 2008).
ROS were measured by the formation of 2,7,-dichlorofluorescein
(DCF). Five microliters of the homogenates were added to 1945 μL of
TRIS-HEPES (18:1) and were incubated in the presence of 50 μL of 2,7-
dichlorofluorescin diacetate (DCFH-DA) for one hour at 37 °C. The re-
action was stopped by freezing. The fluorescence was measured in an
RF5000U Shimadzu Spectrophotometer at 488 nm (excitation) and
525 nm (emission) wavelengths. The results were expressed as ng of
2,7-dichlorofluorescein (DCF) formed/mg protein/h.
To quantify the REDOX environment markers GSH and GSSG, we
used the technique of Hissin and Hilf (1976). Briefly, 300 μL aliquots
were treated with 30% phosphoric acid and were centrifuged at
10,000g for 30 min at 4 °C. To determine GSH, we added 30 μL of the
supernatant, diluted 1:10 with FEDTA (100 mM phosphate and 5 mM
EDTA) in 1.9 ml of FEDTA.
The mixture was reacted with 100 μL of o-phthaldialdehyde. To
measure the GSSG, we took 130 μL of supernatant and added 60 μL of
N-ethylmaleimide. After 30 min, 60 μL of the mixture was mixed into
1.84 ml of FEDTA, and 100 μL of o-phthaldialdehyde was added. The
two chemical species were measured in an RF5000U Shimadzu spec-
trophotometer at 350 nm (excitation) and 420 nm (emission) wave-
lengths.
The results were expressed as ng of GSH or GSSG/mg protein. The
GSH2/GSSG ratio was used as an indicator of the REDOX environment,
as previously described.
2.8. Statistical analysis
For all variables, the results are given as the mean ± SE, and they
were evaluated using one-way analysis of variance and Student-
Newmann-Keuls posthoc test. P < .05 was considered statistically sig-
nificant.
3. Results
The effect of A. maxima and C-phycocyanin administration on 5/6
nephrectomized (5/6 NFx) rats was left ventricular dysfunction, as
observed in Table 1. The 5/6 NFx rats present an increase in systolic
blood pressure (80%) and left ventricular hypertrophy, evidenced by
reductions in left ventricular space (60%) and thickening of the inter-
ventricular septum (50%). The A. maxima and C-phycocyanin admin-
istration, on the other hand, led to a reduction of hypertension. The 5/6
NFx with A. maxima treatment saw an increase in systolic blood pres-
sure by only 30%; left ventricular hypertrophy decreased, as evidenced
by the decrease in left ventricular space by about 16%, and the thick-
ening of the interventricular septum by about 28%. The C-phycocyanin
treatment in the 5/6 NFx reduced cardiovascular alterations more than
the administration of A. maxima, as the systolic blood pressure only
increased by about 8%, and the treatment also prevented left ven-
tricular hypertrophy.
Table 2 shows the effect of A. maxima and C-phycocyanin admin-
istration on some renal function parameters in the 5/6 NFx rats. We
found proteinuria (463%), hyperuricemia (45%), and an increase in
serum creatinine (10%) and its clearance (200%) in the 5/6 NFx rats.
Meanwhile, the treatment with A. maxima and C-phycocyanin partially
prevented alterations in urinary protein, creatinine clearance, and seric
uric acid concentration in the 5/6 NFx rats.
The administration effect of A. maxima and C-phycocyanin on oxi-
dative stress markers can be seen in Fig. 1. The oxidative stress markers
evaluated were the quantification of ROS (ROS, panel A, and panel C)
and lipid peroxidation (LP, panel B, and panel D) in the kidney (panels
A and B) and heart (panels C and D). In the kidney, it was found that the
5/6 NFx rats had an enhanced ROS concentration of 143%, and 88%
lipid peroxidation compared with their respective sham groups.
Meanwhile, the 5/6 NFx rats with A. maxima and C-phycocyanin
treatment saw an increase in ROS concentration of about 78% and 66%,
and they avoided undergoing the lipid peroxidation process. With re-
spect to the heart, the 5/6 NFx rats saw an increase in ROS

39
I.N. Memije-Lazaro et al. Journal of Functional Foods 43 (2018) 37–43

Table 2 kidney except for the 5/6 NFx rats with C-phycocyanin treatment.
Effect of the administration of A. maxima and C-phycocyanin on renal function markers in However, the nephrectomy caused an increase in the GSSG by about
5/6 nephrectomized (5/6 NFx) rats.
145%. Meanwhile, the treatment with A. maxima enhanced the GSSG
Proteinuria Serum creatinine Creatinine Uric acid by about 119%, and the C-phycocyanin did not increase the GSSG
clearance concentration. With regard to the heart, the 5/6 NFx rats saw an in-
mg/dL mg/dL mL/min mg/dL crease in the GSH (121%), GSSG (190%), and GSH2/GSSG index (68%).
However, the 5/6 NFx animals treated with A. maxima or C-phyco-
Sham 3.0 ± 1.4 1.0 ± 0.01 0.5 ± 0.05 4.0 ± 0.4
5/6 NFx 16.9 ± 2.7* 1.1 ± 0.02* 1.5 ± 0.1* 5.8 ± 0.6* cyanin saw only an increase in the GSH (153% for A. maxima and 160%
Sham + A. 1.2 ± 0.9 1.0 ± 0.01 0.4 ± 0.04 3.4 ± 0.6 for C-phycocyanin) and the GSH2/GSSG ratio (762% for A. maxima and
maxima 394% for C-phycocyanin) without enhancing the GSSG.
5/6 NFx + A. 1.2 ± 1.0** 1.2 ± 0.02*,** 0.7 ± 0.2*,** 4.9 ± 0.7*,**
maxima
Sham + C- 1.8 ± 1.2 1.0 ± 0.1 0.6 ± 0.1 3.2 ± 0.6 4. Discussion
phyco-
cyanin
5/6 NFx + C- 3.3 ± 0.2** 1.0 ± 0.04 1.1 ± 0.2*,** 3.8 ± 0.03** The 5/6 nephrectomy (5/6 NFx) is a heuristic model of CKD char-
phyco- acterized by a serum creatinine increase and its clearance, proteinuria,
cyanin and cardiovascular changes such as hypertension and left ventricle
hypertrophy (Sviglerova et al., 2010).
The data represent the mean of six independent data ± SE. One-way ANOVA and
Our results show that the administration of Arthrospira maxima of-
Student-Newman-Keuls post hoc.
* P < .05 vs sham.
fers a partial protection against cardiovascular and renal alterations in
** P < .05 vs 5/6 NFx. NFx rats. However, the C-phycocyanin offers a better cardiorenal axis
protection than Arthrospira maxima at the doses used in this study.
concentration by about 79% without causing lipid peroxidation. How- CKD is characterized by an adaptive and compensatory response of
ever, the 5/6 NFx rats with A. maxima treatment saw an increase in ROS the remaining renal mass to maintain the glomerular filtration rate,
concentration by about 44%, and C-phycocyanin treatment prevented which causes renal afferent arteriolar vasodilatation. Also, CKD causes
the ROS increment. systemic hypertension because of the activation of the renin-angio-
In Fig. 2, the effect of A. maxima and C-phycocyanin administration tensin-aldosterone system (RAAS) (Layton, Edwards, & Vallon, 2017).
on redox environment markers can be observed. We determined glu- During CKD progression, the RAAS is overactivated, and the cardiac
tathione (GSH; panel A and D), oxidized glutathione (GSSG; panel B and work increases. In this way, an adaptive remodeling process in the left
E), and GSH2/GSSG ratio (panel C and F) as redox environment markers ventricle is activated as a long-term mechanism to maintain mean blood
of the kidney (panels A, B, and C) and heart (panels D, E, and F). None pressure, as we can observe in the 5/6 NFx rats (Sayer & Bhat, 2014;
of the treatments modified the GSH or the index GSH2/GSSG in the Tarone & Lembo, 2003).
The nutraceutical action of Arthrospira (Spirulina) and some of its

A C Sham
(ng of DCF formed / mg protein / h)

NFx5/6
(ng of DCF formed / mg protein / h)

1000 2.0
*
*
ROS quantification

800
ROS quantification

, 1.5

* **
, * **
600
**
1.0
400 **
0.5
200

0 0.0

Vehicle A.maxima C-phycocyanin Vehicle A.maxima C-phycocyanin

B D

0.25
0.5
*
Lipid per oxidation

0.4
(RFU / ng protein)

0.20
Lipid per oxidation
(RFU / ng protein)

** ** 0.3
0.15

0.10 0.2

0.05 0.1

0.00 0.0

Vehicle A.maxima C-phycocyanin Vehicle A.maxima C-phycocyanin

Fig. 1. Quantification of reactive oxygen species (panels A and C) and lipid peroxidation (panels B and D) as markers of oxidative stress in the kidney (panels A and B) and heart (panels C
and D) in 5/6 nephrectomized (5/6 NFx) .The data represent the mean of six independent data ± SE. One-way ANOVA and Student-Newman-Keuls post hoc. (*) P < .05 vs sham, (**)
P < .05 vs 5/6 NFx.

40
I.N. Memije-Lazaro et al. Journal of Functional Foods 43 (2018) 37–43

A Sham
D NFx5/6
5 *
100

(μg de GSH / mg protein)

(μg de GSH / mg protein)

4
GSH Content 80

GSH Content
3 *
*
60
2
40
1
20

0
0
Vehicle A.maxima C-phycocyanin
Vehicle A.maxima C-phycocyanin

B E
(μg de GSSG / mg protein)

(μg de GSSG / mg protein)

2.5 15
*
*
2.0
*,**
10
GSSG

GSSG
1.5

1.0 **
5
0.5

0.0 0

Vehicle A.maxima C-phycocyanin Vehicle A.maxima C-phycocyanin

C F
40 1500
**
*,** *,**
GSH2 / GSS Gratio

GSH2 / GSS Gratio

30 1000

500
20
300
*
200
10
100

0 0

Vehicle A.maxima C-phycocyanin Vehicle A.maxima C-phycocyanin

Fig. 2. Determination of the concentration of reduced glutathione (GSH; panel A and D), oxidized glutathione (GSSG; panel B and E) and the radius GSH2/GSSG (panel C and F) as
markers of kidney REDOX environment (panels A, B and C) and heart (panels D, E and F) in 5/6 nephrectomized (5/6 NFx). The data represent the mean of six independent data ± SE.
One-way ANOVA and Student-Newman-Keuls post hoc. (*) P < .05 vs sham, (**) P < .05 vs 5/6 NFx.

metabolites against kidney damage are due to the fact that they act as
an antioxidant and an anti-inflammatory, or they reduce peripheral
vascular resistance among other factors (Fernandez-Rojas et al., 2014;
Rodriguez-Sánchez et al., 2012; Sinanoglu et al., 2012; Zheng et al.,
2013). This is because A. maxima contains compounds such as poly-
phenols, β-carotene, vitamins C and E, polyunsaturated fatty acids ω-3
and ω-6, and phycobiliproteins such as C-phycocyanin (Wu et al.,
2016). Also, the Spirulina diet supplementation could be considered a
functional food because it enhances energy digestibility in the gastro-
intestinal tract that improves the mucosal architecture of the intestine,
leading to better nutrient digestibility and absorption, with beneficial
effects for the organism’s economy (Furbeyre, van Milgen, Mener,
Gloaguen, & Labussiere, 2017). The A. maxima metabolism in the gas-
trointestinal tract could liberate the metabolites mentioned above.
However, the pancreatic and enteric proteases could hydrolyse the
proteins into peptides and oligopeptides. It has been demonstrated that
A. maxima prevents hypertension, cardiac remodeling, and kidney da-
mage because it has two bioactive tripeptides (IQP and VEP) that are
not affected by low gastrointestinal pH or proteases effect and de-
monstrate angiotensin-converting enzyme (ACE) inhibitory activity. In
the kidneys and hearts of spontaneous hypertensive rats (SHR), the
peptides mentioned above cause a down-regulation of the AT1 re-
ceptors and ACE with a reduction in the angiotensin-II synthesis (Pan
et al., 2015). These events cause an anti-hypertensive response, and,
over a long period of time, the renal damage will be delayed and the
cardiac remodeling will be reduced; this is because of an inhibition of
positive feedback loops in the renal mechanism that maintains mean
blood pressure (Ameer et al., 2016; Pan et al., 2015). Also, the peptides
IQP and VEP have a beneficial effect on the kidney because they in-
crease the angiotensin-(1-7) synthesis and the overexpression of an-
giotensin-converting enzyme 2 (ACE2). The binding of Ang-(1-7) to the
AT1 receptor in the kidney produces vasodilatation, natriuresis, and
anti-proliferation responses that have a positive effect reducing CKD
(Iwai & Horiuchi, 2009). Another event that participates in the fibrotic
process in the kidney and heart, causing cardiovascular remodeling, is
oxidative stress (Cristobal-Garcia et al., 2015; Zhang et al., 2007). ROS
has a crucial role in the cellular signaling of cardiac remodeling, as it
promotes vascular smooth-muscle cell growth and migration, en-
dothelial dysfunction and activation, and modification of the extra-
cellular matrix. Also, CKD causes oxidative stress-induced

41
I.N. Memije-Lazaro et al.
proinflammatory cytokines to increase, such as the IL-1β, IL-1 receptor
antagonist, IL-6, TNF-α, C reactive protein, and fibrinogen. The im-
mune system not only secretes these molecules, but the renal resident
cells and adipose tissue participate in the systemic inflammatory pro-
cess (Akchurin & Kaskel, 2015). It has also been observed that CKD
induces profibrotic cascade in the kidney and heart due to an increase
in TGF-β in the kidney (Zhang et al., 2017). Therefore, CKD causes
oxidative stress and inflammation in the kidney and heart and thereby
promotes cardiovascular complications. In this respect, the Arthrospira
(Spirulina) has an antioxidant property that reduces the signaling
pathways of cardiac remodeling because it contains antioxidant mole-
cules such as carotenes, phenolic compounds, and phycobiliproteins,
which reduce the activity of JNK, p38, and NFκB (Wu et al., 2016).
Also, it has been observed that the administration of Spirulina reduces
the synthesis of TNF-α, IL-1β, and IL-6, which also participate in the
remodeling process (Sayer & Bhat, 2014; Tarone & Lembo, 2003). The
C-phycocyanin is one of the metabolites responsible for the action
mentioned above. This protein has a beneficial effect on the cardio-
vascular system because it increases the eNOS expression in the en-
dothelium of large vessels such as the aorta (Ichimura et al., 2013).
Also, it has been found that phycocyanobilin (the prosthetic chromo-
phore group of C-phycocyanin) upregulates the endothelial stress-re-
sponse enzyme heme oxygenase 1 (HMOX-1). The HMOX-1 over-
expression is associated with a reduction in the inflammation, oxidative
stress, apoptosis, and cardiac remodeling (Hu, Chen, Chiang, & Chau,
2004; Strasky et al., 2013). In fact, our results demonstrate a partial
beneficial effect of A. maxima and C-phycocyanin on CKD-related car-
diac and renal oxidative stress. In the kidney, our treatment partially
prevents the increase of oxidative stress markers (LP and ROS), and
only the C-phycocyanin prevents the CKD-related LP and GSSG en-
hancement. Meanwhile, in the hearts of 5/6 NFx rats, our treatments
prevented the CKD-related ROS increase, but the A. maxima and C-
phycocyanin increased the GSH and GSH2/GSSG ratio. The results re-
garding oxidative stress are different in both organs evaluated. It was
observed that the oxidant damage to the kidney was greater than the
damage to the heart, and one reason for this is that the remaining
metabolic function of the kidney is not compensated by the intrinsic
antioxidant compensatory system. Also, 5/6 NFx causes hyperuricemia
and this metabolic state alters the mitochondrial function that causes
oxidative stress, and it maintains systemic hypertension, which leads to
a positive feedback loop in the renal system, causing progressive oxi-
dative stress and renal damage (Cristobal-Garcia et al., 2015). The heart
suffers less oxidative stress than the kidney because because it main-
tains its functionality when we perform the 5/6 nephrectomy, and it
gradually develops an antioxidant compensatory mechanism to prevent
cell damage such as GSH and GSH2/GSSG ratio enhancement. It has
been observed that GSH2/GSSG ratio modification, particularly GSSG
enhancement, causes the S-glutathionylation of proteins, and this pro-
cess is relevant to cellular redox balances and to signaling pathways,
which participate in cardiovascular remodeling (Chen et al., 2010). For
example, GSH reduction in endothelial cells in the kidney promotes the
S-glutathionylation of eNOS, which induces renal fibrosis (Espinosa-
Díez et al., 2018). The S-glutathionylation of the different proteins in
the cardiovascular system that modulate muscular contractility, cellular
growth, metabolism, and inflammation also participates in the re-
modeling process (Pimentel et al., 2012). Thus, it is possible that A.
maxima and C-phycocyanin treatment, as a result of its antioxidant
property, reduces the S-glutathionylation; it has been observed, for
instance, that A. maxima and C-phycocyanin avoid the increase of GSSG
and enhance the GSH2/GSSG ratio, not only in this context but also in
acute kidney injuries (Rodriguez-Sánchez et al., 2012), in doxorubicin-
related cardiotoxic damage, and in other sites of the cardiovascular
system.
Finally, there a difference between the nutraceutical action of
Spirulina and C-phycocyanin on CKD. First, A. maxima (Spirulina)
contains less than 2% C-phycocyanin; therefore, a dose of 1 g/kg of the
42
Journal of Functional Foods 43 (2018) 37–43
cyanobacterium contains about 2 mg/kg of C-phycocyanin. However,
the other components of A. maxima (Spirulina), such as carotenoids,
chlorophylls, and vasoactive peptides, have a synergistic effect that
prevents CKD-related cardiovascular complications, as we have pre-
viously mentioned. With higher doses of Spirulina, we can probably
prevent all cardiovascular complications. However, if we used higher
doses of this cyanobacterium, we would have to modify the bolus
gastrointestinal tract, because not all mammals possess a gut protease
system or microbioma that completely degrades the Spirulina fiber
(Furbeyre et al., 2017). For this reason, we purify the C-phycocyanin,
which is associated with vascular endothelial modulation (Juarez-
Oropeza, Mascher, Torres-Duran, Farias, & Paredes-Carbajal, 2009). We
have proved that a concentrated nutraceutical form of C-phycocyanin
(100 mg/kg of C-phycocyanin) prevented cardiovascular complications
more than Spirulina, because, as previously mentioned, it is a potent
antioxidant that prevents such complications. However, the C-phyco-
cyanin probably has long-term effects, because the C-phycocyanin
metabolism liberates its prosthetic group (phycocyanobilin). The che-
mical similarity between the phycocyanobilin and bilirubin share the
same metabolic pathways, and they probably have a similar half-life of
about 17–20 days (Terry, Maines, & Lagarias, 1993)
The experimental intervention employing Arthrospira (Spirulina)
and C-phycocyanin as a CKD treatment has a nutraceutical effect, of-
fering cardiovascular and renal protection. We conclude that A. maxima
and C-phycocyanin prevent the progression of chronic kidney disease
and its cardiovascular complications, because they have ne-
phroprotective and cardioprotective properties. Also, the C-phyco-
cyanin has the better beneficial effect against CKD-related renal dys-
function and cardiovascular complications compared to A. maxima.
Acknowledgements
This study was partially supported by CONACYT (221057) and
Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional
(20171416, 20171284 and 20171152). We thank INSTITUTO
POLITÉCNICO NACIONAL and CONACyT for financial support. The
researchers are fellows of EDI, COFAA and SNI. The authors have not
interest conflict.
We thanks to Lic. Edgar Salinas and Salomon Shamosh Halabe Ph.D.
from Alimentos Esenciales para la Humanidad S.A. de C.V. for the
Spirulina donation.
We thank John Mitchell for the revision and editing in the English
language.
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