Biomedicine & Pharmacotherapy 100 (2018) 575–582

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

The protective effect of astaxanthin against cisplatin-induced nephrotoxicity T

in rats
⁎
Gorkem Akcaa, Huseyin Erena, , Levent Tumkayab, Tolga Mercantepeb, Mustafa Ozan Horsanalic,
Ezgi Devecid, Eyup Dila, Adnan Yilmaze
a
Recep Tayyip Erdogan University School of Medicine, Urology Department, Rize, Turkey
b
Recep Tayyip Erdogan University School of Medicine, Histology and Embryology Department, Rize, Turkey
c
Recep Tayyip Erdogan University Training and Research Hospital, Urology Department, Rize, Turkey
d
Recep Tayyip Erdogan University School of Medicine, Rize, Turkey
e
Recep Tayyip Erdogan University School of Medicine, Biochemistry Department, Rize, Turkey

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

Keywords: Purpose: The aim of this experimental study was to investigate the antioxidant effects of astaxanthin against
Astaxanthin cisplatin-induced nephrotoxicity in rats.
Antioxidant Methods: Forty-eight male Sprague-Dawley rats weighing 264.83 ± 7.39 g were randomly divided into six
Cisplatin groups of eight animals each. These were constituted as control, olive oil control, astaxanthin control, cisplatin
Nephrotoxicity
control, 16 mg/kg cisplatin & 25 mg/kg astaxanthin and 16 mg/kg cisplatin & 75 mg/kg astaxanthin groups.
Oxidant
Biochemical evaluation was performed by measuring blood urea nitrogen, serum creatinine, total oxidant status
and total antioxidant status. Renal corpuscle, proximal and distal tubules areas (μm2) were calculated for his-
topathological evaluation, and Caspase-3 staining was performed for immunohistochemical evaluation.
Results: Cisplatin reduced total antioxidant status levels and increased blood urea nitrogen, serum creatinine,
total oxidant status, and Caspase-3 levels. It also caused dilatation, vacuolization, and loss of tubular epithelial
cells in the proximal and distal tubules, and glomerular degeneration and edema were determined in kidney
tissue (p < 0.05). Administration of 25 mg and 75 mg astaxanthin increased total antioxidant status levels,
reduced blood urea nitrogen, serum creatinine, total oxidant status, and Caspase-3, and ameliorated degen-
erative distal and proximal tubules, glomerular degeneration and edema in kidney tissue (p < 0.05).
Conclusions: The nephrotoxic effect of cisplatin was diminished by the antioxidant effect of astaxanthin.

1. Introduction damage, apoptosis and necrosis [4,5]. Cisplatin accumulates most in the
S3 segment of the proximal tubules, followed by the distal collecting
Antineoplastic chemotherapeutic agents have been used for many tubules and the S1 segment of the proximal tubules [6].
years, but because these agents disrupt the cell cycle, both normal and Exposure to oxidant radicals is increasing steadily in industrialised
neoplastic cells are affected by them [1]. Cisplatin is a platinum-based societies. Oxidative stress plays an active role in acute kidney injury
drug and a useful chemotherapeutic agent in various malignancies, induced by cisplatin administration. Cisplatin specifically increases the
including solid malignant tumours of the head, neck, bladder, testis, production of hydroxyl radicals, potent free radicals that consume in-
ovary, prostate, cervix, oesophagus and lung [2]. Cisplatin has several tracellular antioxidant stores and affect directly cell components, such
side effects, such as nausea and vomiting, nephrotoxicity, neurotoxicity, as lipids, proteins and DNA, thus impairing the cellular structure [7,8].
ototoxicity and, rarely, ocular toxicity [3]. Nephrotoxicity is the most Reactive oxygen species (ROS) involved in cisplatin nephrotoxicity in-
frequent and dose-limiting side effect of cisplatin therapy, and irre- clude superoxide anion, hydrogen peroxide, hydroxyl radical and re-
versible kidney damage occurs in one-third of patients despite protec- active nitrogen species, such as peroxynitrite and nitric oxide [9–11].
tive measures [1,2]. Nephrotoxic effects occur in several ways, in- Cisplatin also inhibits antioxidant enzymes; hence, the renal activities
cluding via oxidative stress, inflammation, fibrogenesis, mitochondrial of superoxide dismutase (SOD), glutathione peroxidase (GPx),

⁎
Corresponding author.
E-mail addresses: [email protected] (G. Akca), [email protected] (H. Eren), [email protected] (L. Tumkaya),
[email protected] (T. Mercantepe), [email protected] (M.O. Horsanali), [email protected] (E. Deveci), [email protected] (E. Dil),
[email protected] (A. Yilmaz).

https://doi.org/10.1016/j.biopha.2018.02.042
Received 1 January 2018; Received in revised form 11 February 2018; Accepted 13 February 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
G. Akca et al. Biomedicine & Pharmacotherapy 100 (2018) 575–582

glutathione reductase, total antioxidant status (TAS) and catalase de-

crease markedly [12,13]. Malondialdehyde (MDA) and total oxidant
status (TOS) levels also increase as a result of damage to antioxidant
systems [13–15]. TAS is an effective assay in both human and animal
models for the measurement of serum antioxidative stress [16,17]. Its
use as an antioxidant in the literature is increasing steadily [18]. TAS
and TOS are also easy-to-perform, stable, reliable, sensitive and in-
expensive methods for measuring total antioxidant capacity [19].
Carotenoids, an antioxidant group, include over 700 organic, so-
luble substances produced by phytoplankton, algae, plants and limited
numbers of fungi and bacteria [20]. Astaxanthin is a highly antioxidant,
anti-inflammatory, carotenoid pigment, and it is considered the stron-
gest and safest known antioxidant in nature. It is obtained from the
microalgae Haematococcus pluvialis [21]. Astaxanthin exhibits anti- Fig. 1. Schematic experimental timeline.

oxidant activities through two pathways: it scavenges free radicals and

protects against chain reactions that may be caused by free radicals or it was the 16 mg/kg cisplatin and 75 mg/kg astaxanthin group (n = 8),
terminates chain reactions that may already have taken place [22]. Due having received 75 mg/kg astaxanthin intraperitoneally daily for eight
to its beneficial effects on human health, astaxanthin is widely used as a days and a single 16 mg/kg dose of cisplatin intraperitoneally on the
nutritional supplement and antioxidant [23,24]. The U.S. Food and fifth day (Fig. 1).
Drug Administration (FDA) approves the use of astaxanthin as a human
dietary supplement. 2.2. Biochemical analysis procedure
This study employed biochemical, histopathological and im-
munohistochemical methods to investigate the antioxidant effects of The kidneys were removed from the abdominal cavities. Phosphate
astaxanthin against cisplatin nephrotoxicity under experimental con- buffered saline PBS), a water-based salt solution containing sodium
ditions. phosphate dibasic, sodium chloride and sodium phosphate monobasic,
was then prepared for kidney tissues (pH: 7.4). The specimens were
2. Materials and methods washed in ice-cold PBS and weighed. The ratio of tissue weight to
homogenisation buffer was 1:10. Specimens were homogenised in PBS
Forty-eight male Sprague Dawley rats aged 3–5 months and for one min. The homogenates were centrifuged at 2717 g for 20 min.
weighing 264.83 ± 7.39 g were procured from the Recep Tayyip TAS and TOS levels were measured using a commercial kit (RL0024,
Erdogan University Animal Care and Research Unit (Rize, Turkey). All Rel Assay Diagnostics, Turkey). The TAS results were expressed as
animals received care according to the criteria outlined in the ‘Guide for Trolox Equivalent/L and the TOS results as μmol H2O2 Equivalent/L.
the Care and Use of Laboratory Animals’ prepared by the National Blood samples were collected from all rats for biochemical evalua-
Academy of Sciences and published by the National Institutes of Health. tion. Blood was separated by centrifugation at 1739 g for 15 min at 4 °C.
Approval for the study was granted by the Recep Tayyip Erdogan Levels of blood urea nitrogen (BUN) and serum creatinine were de-
University (Rize, Turkey) Animal Ethical Committee (No. 2016/32). termined using a standard AutoAnalyzer (Architect c16000
AutoAnalyzer, Abbott Diagnostics, Waltham, Massachusetts, USA). The
2.1. Study design results were expressed as mg/dL [28–30].

Animals were kept in Recep Tayyip Erdogan University Animal Care

2.3. Histopathological analysis procedure
and Research Unit (Rize, Turkey). All animals were maintained and fed
in a sterile experimental animal unit environment having 55–60% hu-
Kidneys were fixed in 10% neutral formaldehyde. Specimens were
midity, at a temperature of 22 ± 3 °C, and 12-h light:12-h light-dark
then dehydrated in an ascending alcohol series. Next, all sections were
cycle. Rats were allowed ad libitum access to commercially available
cleared in xylene and embedded in paraffin using routine laboratory
standard rat chow (Bayramoğlu Feed and Flour Industry Trading
methods. They were stained with hematoxylin and eosin (H&E) and
Corporation Erzurum, Turkey) and tap water throughout the experi-
analysed by two histologists blinded to the study groups under a light
ment. After sufficient time had been allowed for adaptation to the la-
microscope (Leica DM6200-Germany). Photographs were taken with an
boratory conditions, the experimental animals were divided randomly
Olympus DP20 camera.
into six groups.
Anesthesia was administered to all groups with 50 mg/kg in-
traperitoneal ketamine hydrochloride (Ketalar ®, Eczacibası Parke- 2.4. Quantitative analysis
Davis, Istanbul, Turkey) and 10 mg/kg intraperitoneal Xylazine HCl
(Alfazyne ®, Alfasan International BV Woerden, Holland). The renal corpuscle, proximal and distal tubule areas (μm2) were
All groups received oral distilled water daily for eight days. Group 1, measured using the Olympus DP2-BSW (Ver.2.1 to Ver.2.2, Build 6212,
the control group (n = 8), received no drug injections except for an- Olympus Corporation, Tokyo, Japan) software system. This consists of a
aesthetics. Group 2 acted as the olive oil control group (n = 8). Olive oil camera (Olympus DP20, Olympus Corporation, Tokyo, Japan) attached
was used for dissolving astaxanthin in Groups 3–6. Group 2 received to a light microscope (Leica DM6200, Germany) and a computer with a
intra-peritoneal olive oil only for eight days [25]. Group 3, the astax- software system. H&E (Darmstadt, Germany)-stained sections were
anthin control group (n = 8), received only intra-peritoneal astaxanthin placed onto the microscope tray, and their sectional boundaries were
75 mg/kg dissolved in olive oil [26]. Group 4 acted as the cisplatin only determined using this programme (40x objective lens). Once the area
group (n = 8) and received a single 16 mg/kg dose of cisplatin (Cis- was identified, distinct and separate frames were assessed by two
platin DBL 100 mg/100 ml vial, Orna Ilac, Istanbul) intraperitoneally blinded histopathologists (Fig. 2).
on the fifth day of the study [27]. Group 5 represented the 16 mg/kg
cisplatin and 25 mg/kg astaxanthin group (n = 8), having received 2.5. Immunohistochemistry (IHC) analysis procedure
25 mg/kg astaxanthin intraperitoneally daily for eight days and a single
16 mg/kg dose of cisplatin intraperitoneally on the fifth day. Group 6 Sections were deparaffinised and treated with proteinase K solution

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Table 1
Kidney tissue biochemical results.

Group Kidney

TAS TOS

Control 8.69 ± 0.19** 26.07 ± 0.71**

Olive oil 9.16 ± 0.15** 24.81 ± 0.4**
Astaxanthin 75 mg 12.60 ± 0.7** 25.06 ± 0.50**
Cisplatin 6.92 ± 0.75* 34.13 ± 0.88*
Cisplatin + Astaxanthin 25 mg 10.47 ± 0.82** 23.02 ± 0.92**
Cisplatin + Astaxanthin 75 mg 11.13 ± 0.34** 22.20 ± 0.78**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

3. Results

3.1. Biochemical results

2
Fig. 2. The renal corpuscle, proximal and distal tubule areas (μm ) were measured using
the Olympus DP2-BSW (Ver.2.1 to Ver.2.2, Build 6212, Olympus Corporation, Tokyo, TAS and TOS were used as markers for demonstrating the anti-
Japan) software system. oxidant effect of astaxanthin and the oxidative effect of cisplatin, re-
spectively. Following cisplatin administration, we observed increased
(20 μg/mL in PBS, Abcam, UK), washed with distilled water and placed TOS levels and decreased TAS levels (p < 0.05). Astaxanthin 75 mg
in 3% hydrogen peroxide. After several washes with PBS (10x, pH 6.0, alone did not affect TOS levels, but it did increase TAS levels
ab128983, Abcam, UK), the sections were placed into an equilibration (p < 0.05). While a single dose of cisplatin lowered TAS and aug-
buffer. Sections were incubated with Caspase-3 (1:200, Abcam Rabbit mented TOS concentrations, a combination of cisplatin and astaxanthin
polyclonal to active Caspase-3, Abcam, UK). After washing with PBS, all increased TAS levels and reduced TOS levels (p < 0.05) (Table 1)
sections were incubated in a secondary antibody (Rabbit Specific HRP/ (Fig. 3).
DAB [ABC] Detection IHC kit, ab64218, Abcam, UK) and subsequently Cisplatin treatment increased BUN and serum creatinine levels
incubated in anti-digoxigenin-peroxidase. The reaction was revealed (p < 0.05) (Table 2) (Fig. 3), and the astaxanthin-treated groups ex-
with 0.06% 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical, hibited decreased BUN and serum creatinine levels (p < 0.05)
St. Louis, MO) in PBS. Finally, all sections were counterstained with (Table 2) (Fig. 3).
Harris hematoxylin.
3.2. Histopathological results

2.6. Stereological analysis 3.2.1. Light microscopy results

Light microscopic sections from the renal tissue of rats from the
Mean numerical Caspase-3-positive cell densities were calculated control group exhibited regular Bowman’s capsules with normal his-
using the fractional method, a stereological technique involving an tological structural features (Fig. 4A and B). The olive oil and astax-
unbiased counting frame. The Stereo Investigator (MicroBrightField anthin 75 mg groups also exhibited normal tubules, and their Bowman’s
9.0, Colchester, VT, CA, USA) software system was used. This system capsules had regular margins with no additional pathology (Fig. 4C–F).
consists of a camera attached to a light microscope, a motorised system On the other hand, light microscopic sections from the cisplatin group
equipped with a microscope tray and a computer with a software exhibited damage in the renal cortex and medulla (Fig. 4G). We also
system. IHC-Caspase-3-stained sections were placed on the microscope observed the loss of tubular epithelial cells and common tubular dila-
tray, and their sectional boundaries were determined using this pro- tations (Fig. 4H). Furthermore, we detected a cast formation in the
gramme. After determining the area, separate and distinct frames were proximal and distal tubules, as well as capillary congestions in the
identified by systematic random sampling of the sections, in line with peritubular areas (Fig. 4G–H). In the cisplatin + astaxanthin 25 mg-
the rules of space fragmentation with the step interval of the x- and y- treated group, the Bowman’s capsules and proximal and distal tubules
axes. Measurements in 20 different selected areas in all groups were exhibited regular morphologies. Levels of glomerular degeneration and
taken following the method described by Mercantepe [31]. tubular epithelial cell injury were also lower than those of the cisplatin
group (Fig. 4I and J). The cisplatin + astaxanthin 75 mg-treated group
also exhibited regular Bowman’s capsules and proximal and distal tu-
2.7. Statistical analysis bules, similar to those of the astaxanthin 25 mg-treated group (Fig. 4K
and L).
Data were analysed using the SPSS Statistics 20.0 (IBM Inc.,
Chicago, USA) software for statistical calculations. Biochemical vari- 3.2.2. Immunohistochemical (IHC) results
ables were expressed as mean ± standard deviation. Differences be- Sections from the control, olive oil only and astaxanthin 75 mg
tween the groups were tested using the one-way analysis of variance groups were Caspase-3 negative (Fig. 5A–F). However, we observed
(ANOVA) followed by the Tukey HSD test; p values < 0.05 were con- greater Caspase-3 staining of proximal and distal tubular epithelial cells
sidered statistically significant. The renal corpuscle, proximal and distal in the cisplatin group compared to the control group (p < 0.05)
tubule areas (μm2) and Caspase-3-positive cell numerical density values (Table 3) (Fig. 5G and H). The astaxanthin 25 mg and astaxanthin
were expressed as the mean ± standard deviation, and differences 75 mg groups exhibited less staining for the Caspase-3 expression than
between the groups were tested using the one-way ANOVA followed by the cisplatin only group (p < 0.05) (Table 3) (Fig. 5I–L).
the Duncan test; p values < 0.05 were regarded as significant.
3.2.3. Semi-quantitative analysis results
Higher Bowman’s capsule and proximal and distal tubular area

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Fig. 3. Biochemical analysis graphs.

A: Total Antioxidan Status. B: Total Oxidan Status. C:Blood Urea Nitrogen. D: Serum Creatinine. *P < 0.05, #P < 0.05; Control vs Cisplatin group, ##
P < 0.05 Cis + Asta 25 mg vs
Cisplatin group, ###P < 0.05 Cis + Asta 75 mg vs Cisplatin group.

Table 2 first to demonstrate nephrotoxicity using cisplatin and second to pre-

Serum biochemical parameters. vent this effect using astaxanthin, an antioxidant agent. Although cis-
platin treatment increased BUN, serum creatinine and TOS levels and
Group
lowered TAS levels, 25 mg and 75 astaxanthin treatment combined with
Bun (mg/dL) Creatinine (mg/dL) cisplatin reduced BUN, serum creatinine and TOS levels and increased
**
TAS levels. There were no statistically significant differences between
Control 40.14 ± 1.95 0.412 ± 0.016**
the 25 mg and 75 mg astaxanthin treatment groups in terms of BUN,
Olive oil 39.42 ± 2.63** 0.395 ± 0.263**
Astaxanthin 75 mg 42.14 ± 1.95** 0.424 ± 0.019** serum creatinine, TAS or TOS concentrations. These biochemical results
Cisplatin 44.28 ± 1.88b* 0.451 ± 0.02* show that astaxanthin protects the kidneys from cisplatin-induced ne-
Cisplatin + Astaxanthin 25 mg 38.57 ± 1.71** 0.394 ± 0.02** phrotoxicity.
Cisplatin + Astaxanthin 75 mg 36.42 ± 1.98** 0.380 ± 0.023** Cisplatin is widely used for the tumours of several organs, such as
the head-neck region, bladder, testis, ovary, prostate, cervix and lung
* p ˂ 0.05 when compared to the control group.
** p ˂ 0.05 when compared to the cisplatin group.
[1,33]. Frequent use results in severe adverse effects, such as ne-
phrotoxicity, neurotoxicity, ototoxicity and, rarely, ocular toxicity [34].
measurements were found in the cisplatin group compared to the Nephrotoxicity, the most common side effect limiting cisplatin dosages,
control group (p < 0.05). The cisplatin + astaxanthin 25 mg and the is observed in almost 30% of patients [35,36]. Due to its low molecular
cisplatin + astaxanthin 75 mg groups exhibited smaller Bowman’s weight, cisplatin permeates the glomerular basal membrane easily and
capsule and proximal and distal tubular area measurements than the accumulates in the proximal tubular inner medulla and outer cortices
cisplatin only group (p < 0.05) (Tables 4–6). [37]. Pabla et al. reported a reduced glomerular filtration rate, in-
creased serum creatinine and decreased magnesium and potassium le-
4. Discussion vels [38]. The pathophysiological effect of cisplatin on the kidneys is
exerted in the form of apoptosis and necrosis in proximal tubule cells
Several studies have investigated the effect of antioxidants on ne- via oxidative stress, inflammation and vasoconstriction of the renal
phrotoxicity [30,32]. However, to the best of our knowledge this is the vascular structure [39,40]. Tubular cell death was identified as the
first experimental study to show the antioxidant effect of astaxanthin on main underlying histopathological characteristic of nephrotoxicity in
cisplatin-induced nephrotoxicity. The main purposes of this study were one experimental study [41]. Histopathological studies have

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Fig. 4. Light microscopic photographs from kidney tissue sections stained with H&
E.
A (x200)-B (x00): Control Group; Healthy kidney tissue from the control group.
Glomerules (GL) and Bowman’s capsules (bs) exhibit regular structures. The prox-
imal (ps) and distal tubules (dt) are typical. Mesangial cells (green arrow) also
exhibit a normal structure. A normal proximal tubule epithelium cell with a brush
border (blue arrow). C (x40)-D (x400): Olive oil Group; Glomerules (GL).
Bowman’s capsule space (bs) with a regular structure. The proximal (ps) and distal
tubules (dt) are typical. Mesangial cells (green arrow) exhibit a normal structure. A
normal proximal tubule epithelium cell with a brush border (blue arrow). E (x200)-
F (x400): Astaxanthin 75 mg Group; Glomerules (GL). A regular Bowman’s cap-
sule space (bs). The proximal (ps) and distal tubules (dt) are typical. Mesangial cells
(green arrow) exhibit a normal structure. A normal proximal tubule epithelium cell
with a brush border (blue arrow). G (x40)-H (x400): Cisplatin Group; Damaged
kidney tissue from the cisplatin group. Degenerative glomeruli (arrow) and glo-
merular amyloidosis (green arrow) can be seen. Necrotic tubular cells and frequent
tubular dilatations are present (comet arrow), together with a tubular cyst forma-
tion (arrowhead). Significantly damaged cells can be seen in the tubules (spiral
arrow). Peritubules can be seen in congenital capillaries. I (x200)-J (x400):
Cisplatin + Astaxanthin 25 mg Group; Glomerulus with a regular structure (gl).
The proximal (pt) and distal tubules (dt) are typical. Bowman’s capsule (arrow). K
(x100)-L (x400): Cisplatin + Astaxanthin 75 mg Group; Glomerulus with a reg-
ular structure (gl). The proximal (pt) and distal tubules (dt) are have a stranded
structure. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

demonstrated interstitial haemorrhage, atrophy, interstitial inflamma- of tubular cells, tubular dilatations and peritubular capillary conges-
tion and tubular dilatation [42,43]. Our histopathological findings were tions in the cisplatin group. In terms of histopathological features, both
similar among the control, olive oil and astaxanthin 75 mg groups. In the cisplatin + astaxanthin 25 mg and cisplatin + astaxanthin 75 mg
contrast, we observed oxidative damage in the cortex and medulla, loss groups exhibited a protective effect against cisplatin-induced toxicity

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Fig. 5. Light microscope image of kidney tissue by light microscopy. Caspase-3 staining.
A (x200)-B (x400): Control Group; Healthy morphological structures in the kidneys. Caspase-3-negative proximal and distal tubular epithelial cells (arrow). C (x200)-D (x400): Olive Oil
Group; E (x200)-F (x400): Astaxanthin 75 mg Group; G (x20)-H (x400): Cisplatin Group; Samples exhibit proximal and distal tubular epithelial cell positivity (arrow). I (x200)-J
(x400): Cisplatin + Astaxanthin 25 mg Group; Proximal and distal tubular epithelial cells (arrow) are immunologically negative. K (x200)-L (x400): Cisplatin + Astaxanthin 75 mg
Group; Proximal and distal tubular epithelial cells (arrow) are immunologically negative.

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Table 3
Caspase-3 positive nuemarical density measurement (mm3) data.

Group Caspase-3 positive nuemarical density measurement (mm3) data (Mean ± Standart Deviation)

Control 3.17 ± 1.72**

Olive oil 2.5 ± 1.64**
Astaxanthin 75 mg 7.67 ± 3.01**
Cisplatin 110.33 ± 12.58*
Cisplatin + Astaxanthin 25 mg 15.00 ± 4.73**
Cisplatin + Astaxanthin 75 mg 15.17 ± 6.27**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

Table 4
Renal corpuscle area measurement (μm2) data.

Group Renal corpuscle area measurement (Mean ± Standart Deviation)

Control 4840.15 ± 676.28**

Olive oil 4986.76 ± 551.44**
Astaxanthin 75 mg 5093.14 ± 568.26**
Cisplatin 10414.61 ± 2712.56*
Cisplatin + Astaxanthin 25 mg 4373.14 ± 904.04**
Cisplatin + Astaxanthin 75 mg 4935.31 ± 154.165**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

Table 5
Proximal tubules area measurement (μm2) data.

Group Proximal tubules area measurement (μm2) data (Mean ± Standart Deviation)

Control 1466.16 ± 153.49**

Olive oil 1591.28 ± 256.01**
Astaxanthin 75 mg 1364.83 ± 256.00**
Cisplatin 3452.18 ± 1058.12*
Cisplatin + Astaxanthin 25 mg 1496.54 ± 123.32**
Cisplatin + Astaxanthin 75 mg 1701.1723 ± 427.15**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

Table 6
Distal tubules area measurement (μm2) data.

Group Distal tubules area measurement (μm2) data (Mean ± Standart Deviation)

Control 2492.52 ± 344.78**

Olive oil 2362.15 ± 359.304**
Astaxanthin 75 mg 2299.48 ± 322.09**
Cisplatin 4798.49 ± 1848.47*
Cisplatin + Astaxanthin 25 mg 1508.07 ± 311.16**
Cisplatin + Astaxanthin 75 mg 2255.53 ± 459.22**

* p ˂ 0.05 versus to control group.

** p ˂ 0.05 versus to cisplatin group.

compared to the cisplatin only group. anti-carcinogenic, anti-angiogenic, neuroprotective, im-

Although the mechanisms underlying the nephrotoxic effects of munomodulator, anti-diabetic and anti-obesity activities [47–49]. Yeh
cisplatin have not yet been elucidated fully, oxidative stress seems to et al. showed that astaxanthin reduces retinal oxidative stress in
play a critical role in oxidative damage, while hypoxia and mitochon- streptozocin-induced diabetic rats [50]. Similarly, Mosaad reported
drial damage are implicated in the pathogenesis of cisplatin ne- that astaxanthin protects the kidneys from gentamicin-induced ne-
phrotoxicity [44]. Previous studies have observed indicators of renal phrotoxicity [51].
oxidative damage, such as increased MDA and TOS and reduced glu- Faubel et al. reported that cisplatin-induced nephrotoxicity causes
tathione (GSH), TAS and SOD levels in cisplatin-induced ne- apoptosis in renal tubular epithelial cells, in addition to acute tubular
phrotoxicities [13,45]. Verma described both TAS and TOS as useful necrosis [52]. Caspase-3 activation is regarded as an irreversible
assays for evaluating oxidative stress [13]. Ali showed in a review study terminal event before cell death, and it is used as a marker of apoptosis
that oxidative damage plays an essential role in the pathogenesis of [52]. In our study, we also determined that cisplatin causes apoptosis in
nephrotoxicity, as well as reported that a large number of antioxidant renal tubular epithelial cells (p < 0.05) (Table 3) (Fig. 5G and H).
agents has been investigated in terms of prevention [46]. Astaxanthin, a Another experimental study showed that astaxanthin reduces oxidative
carotenoid pigment obtained from the microalgae Haematococcus plu- stress via cytochrome C, Caspase-9 and Caspase-3 in early acute renal
vialis, is a potent antioxidant with anti-inflammatory, anti-apoptotic, failure [53]. Similarly, we determined that astaxanthin reduces the

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