Chapter 8

Effect of Astaxanthin on Antioxidant Status,

Histology and Calcium Deposition in Renal Tissue

The lipid peroxidation and antioxidant status in the renal tissue of the
experimental animals were monitored to investigate the effect of astaxanthin
on renal damage caused by calcium oxalate crystal induced oxidative stress.
The level of lipid peroxidation was measured by measuring the level of
thiobarbituric acid reactive substances (TBARS). The activities of the
enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase
(GPx), glutathioneS-transferase (GST) and glutathione reductase (GR) were
determined. The non enzymatic antioxidants: reduced glutathione (GSH),
vitamin C and vitamin E were also assessed. These variables give
information to the degree of oxidative stress experienced by an organism and
its ability to recover. The effect of astaxanthin on renal histology and
calcium oxalate deposition was also studied through haematoxylin and eosin
(H&E) staining, Pizzolato staining, flame photometry and scanning electron
microscopy coupled with energy dispersive X-ray (SEM-EDX) analysis. All
the above parameters were also investigated on nephrolithiatic animals
treated with potassium citrate.
 8.1 Results
8.1.1. Effect of astaxanthin on lipid peroxidation in kidney

194
Figure 8.1 Thiobarbituric acid reactive substances (TBARS)-kidney
C(21D) and C(42D) are the 21 and 42 days controls, Asx-25(21D)C, Asx-25(42D)C, Asx-50(21D)C and Asx-50(42D)C are groups treated with
astaxanthin 25 mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL is the nephrolithiatic
group. NPL+ Asx-25(21D), NPL+ Asx-25(21D), NPL+ Asx-25(42D) and NPL+ Asx-50(21D) are nephrolithiatic groups treated with astaxanthin 25
mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL+ Asx- CIT (21D) and NPL+ CIT (42D)
are nephrolithiatic groups treated with citrate for 21 and 42 days.

Chapter 8
Data are represented as mean SD for six observations. * represents significant difference from all the control groups, represents significant
difference from the nephrolithiatic group and represents significant difference from all the 25 mg/kg b.wt. astaxanthin treated for 21 days group.
p<0.05 was considered significant.
Effect of Astaxanthin on Antioxidant Status, Histology and 195

The extent of lipid peroxidation was assessed in the kidney of the

experimental animals and the results are given in Figure 8.1. A significantly
high level of lipid peroxidation was observed in the renal tissue of
nephrolithiatic animals when compared to the controls (p<0.05). TBARS
levels remained high in the 21 days treated group administered with
25mg/kg b.wt. astaxanthin than all the other treated groups. On the contrary,
all other treatments with astaxanthin significantly reduced renal lipid
peroxidation when compared to that of the nephrolithiatic animals (p<0.05)
with no significant differences among the treated groups.

The citrate treated groups at both the durations had TBARS levels
significantly lesser than the nephrolithiatic groups and the 25 mg/kg b.wt for
21 days astaxanthin administered group (p<0.05).
8.1.2. Effect of astaxanthin on renal antioxidant system
8.1.2.1. Effect of astaxanthin on kidney superoxide dismutase

196
Figure 8.2 Superoxide dismutase- Kidney
C(21D) and C(42D) are the 21 and 42 days controls, Asx-25(21D)C, Asx-25(42D)C, Asx-50(21D)C and Asx-50(42D)C are groups treated with
astaxanthin 25 mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL is the nephrolithiatic
group. NPL+ Asx-25(21D), NPL+ Asx-25(21D), NPL+ Asx-25(42D) and NPL+ Asx-50(21D) are nephrolithiatic groups treated with
astaxanthin 25 mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL+ Asx- CIT (21D) and
NPL+ CIT (42D) are nephrolithiatic groups treated with citrate for 21 and 42 days. Data are represented as mean SD for six observations. *

Chapter 8
represents significant difference from all the control groups, represents significant difference from the nephrolithiatic group, # represents
significant difference from the 25 mg/kg b.wt. astaxanthin treated for 42 days group, represents significant difference from both the 25
mg/kg b.wt. and 50 mg/kg b.wt. astaxanthin treated for 21 days groups and represents significant difference from all the control groups and
astaxanthin treated groups. p<0.05 was considered significant.
Effect of Astaxanthin on Antioxidant Status, Histology and 197

Figure 8.2 demonstrates the SOD activity in the kidney of the

experimental animals. It was seen that the SOD activity was significantly
diminished in the nephrolithiatic animals (p<0.05). Treatment of the
nephrolithiatic animals with astaxanthin brought about an elevation in the
SOD levels at all doses and durations but the SOD levels did not reach the
normal levels. The group treated with 50 mg/kg b.wt. astaxanthin for 42 days
had significantly higher SOD values than all other treated groups (p<0.05).
These values were similar to the control groups.

Citrate treatment for both 21 and 42 days did not bring about any
significant change in the SOD values from the nephrolithiatic group. Both
the duration treatments had significantly low levels of the SOD enzyme than
all the astaxanthin treated and control groups (p<0.05).
 8.1.2.2. Effect of astaxanthin on kidney catalase

198
Figure 8.3 Catalase - Kidney
C(21D) and C(42D) are the 21 and 42 days controls, Asx-25(21D)C, Asx-25(42D)C, Asx-50(21D)C and Asx-50(42D)C are groups treated with astaxanthin
25mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL is the nephrolithiatic group. NPL+ Asx-25(21D),
NPL+ Asx-25(21D), NPL+ Asx-25(42D) and NPL+ Asx-50(21D) are nephrolithiatic groups treated with astaxanthin 25 mg/kg b.wt. for 21 and 42 days and
astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL+ Asx- CIT (21D) and NPL+ CIT (42D) are nephrolithiatic groups treated with citrate for
21 and 42 days.
Data are represented as mean SD for six observations. * represents significant difference from all the control groups, represents significant difference from
the nephrolithiatic group and, $ represents significant difference from the controls and astaxanthin

Chapter 8
25 mg/kg b.wt controls, represents significant difference from the 50mg/ kg b.wt. astaxanthin treated groups, represents significant difference from all the
25 mg/ kg b.wt. astaxanthin treated for 21 days group, represents significant difference from all the 25 mg/ kg b.wt. astaxanthin treated groups and
represents significant difference from all the control groups and astaxanthin treated groups. p<0.05 was considered significant.
Effect of Astaxanthin on Antioxidant Status, Histology and 199

The catalase activity is reduced significantly in the kidney of the

nephrolithiatic animals (Figure 8.3). There is an induction in the catalase
activity in the 50 mg/kg b.wt. astaxanthin controls in the 21 and 42 days
groups which is significant (p<0.05). All the treatments increased the
catalase values when compared to the nephrolithiatic group. But the 21 days
group treated with 25 mg/kg b.wt. astaxanthin the catalase activity remained
low. In contrast, all the other treated groups showed a significantly higher
enzyme activity with the 50 mg/kg b.wt. for 42 days showing maximum
effect (p<0.05).

The citrate treated groups exhibited catalase levels which were

significantly higher than the nephrolithiatic group but lesser than all the other
groups (p<0.05).
 200 Chapter 8

8.1.2.3. Effect of astaxanthin on Glutathione peroxidase (GPx),

Glutathione S-Transferase (GST) and Glutathione reductase
(GR) - Kidney
Table 8.1 Glutathione peroxidase (GPx), GlutathioneS-Transferase
(GST) and Glutathione reductase (GR) - Kidney
GST(mol of GR(nmol of
GPx (g of GSH
CDNB NADPH
GROUPS consumed/min/
formed/min/ mg oxidised/min/ mg
mg protein)
protein) /protein)
Control-21 days 8.450.41 24.902.03 64.274.01
Control-42 days 8.390.20 23.791.98 63.993.97
Astaxanthin 25 mg/kg b.wt.- 21 days
9.571.494 25.482.20 64.392.09
control
Astaxanthin 25 mg/kg b.wt.- 42 days
9.442.03 27.870.71 65.433.21
control
Astaxanthin 50 mg/kg b.wt.- 21 days
10.891.47 c 34.592.98$ 67.983.33
control
Astaxanthin 50 mg/kg b.wt.- 42 days
12.091.20 c 32.091.76$ 69.005.50
control
Nephrolithiatic 4.790.13* 15.610.71* 42.362.90*
Nephrolithiatic treated astaxanthin 25
6.230.13* 18.890.45* 54.603.96*
mg/kg b.wt.-21 days
Nephrolithiatic treated astaxanthin 25
6.990.43* 22.091.54 62.921.96
mg/kg b.wt.-42 days
Nephrolithiatic treated astaxanthin 50
8.580.57 23.702.04 53.985.32*
mg/kg b.wt.-21 days
Nephrolithiatic treated astaxanthin 50
11.980.88 c 26.091.09 65.004.89
mg/kg b.wt.-42 days
Nephrolithiatic treated with citrate-
3.570.21 18.70.18 49.274.01
21 days
Nephrolithiatic treated with citrate-
4.330.32 19.221.04 50.392.09
42 days

Data are represented as mean SD for six observations. * represents significant

difference from all the control groups, represents significant difference from the
nephrolithiatic group and, c represents significant difference from the controls,
represents significant difference from the 50 mg/kg b.wt. astaxanthin treated groups,
represents significant difference from a the 25 mg/kg b.wt. astaxanthin treated for 21
days group, represents significant difference from both the 25 and 50 mg/kg b.wt.
astaxanthin treated for 21 days groups and represents significant difference from all
the control groups and astaxanthin treated groups. p<0.05 was considered significant.
Effect of Astaxanthin on Antioxidant Status, Histology and 201

Table 8.1 delineates the activities of the renal antioxidant enzymes

GPx, GST and GR in the experiment. The nephrolithiatic animals showed a
marked decrease compared to the controls in all the three enzyme levels
assessed. These depleted enzyme levels were found to be normalized on
astaxanthin treatment. The control groups fed with astaxanthin at
50 mg/kg b.wt. exhibited an increase in the activities of the GPx, GST and
GR enzymes when compared to the normal controls. The increase was
significant in the case of GPx and GST (p<0.05).

In case of GPx, the treatment with astaxanthin at 25 mg/kg.b.wt at

both the durations did not bring the enzyme activities to the normal levels.
The GPx activities were increased in the 50 mg/kg b.wt. administered
animals and the values of the 42 days treated animals at this dose were
significantly higher than all the control groups and all the astaxanthin
treated groups (p<0.05). A similar observation was found in the case of
GST also where the 50 mg/kg b.wt. astaxanthin for 42 days showed
significantly more restorative effect than all other treatments. However, in
case of GR, both the 25 and 50 mg/kg b.wt. treatments showed significant
increase at 42 days (p<0.05).

Treatment of the nephrolithiatic animals with citrate did not restore

the levels of the GPx enzyme to normal at both the durations. On the other
hand both the duration citrate treatments increased the levels of GST and GR
significantly when compared to the nephrolithiatic group but at the same
time these values were also significantly lower than all the astaxanthin
treated groups (p<0.05).
8.1.2.4. Effect of astaxanthin on Reduced glutathione - kidney

202
Figure 8.4 Reduced glutathione (GSH) - Kidney
C(21D) and C(42D) are the 21 and 42 days controls, Asx-25(21D)C, Asx-25(42D)C, Asx-50(21D)C and Asx-50(42D)C are groups treated with astaxanthin
25mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL is the nephrolithiatic group. NPL+ Asx-25(21D),
NPL+ Asx-25(21D), NPL+ Asx-25(42D) and NPL+ Asx-50(21D) are nephrolithiatic groups treated with astaxanthin 25 mg/kg b.wt. for 21 and 42 days and
astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL+ Asx- CIT (21D) and NPL+ CIT (42D) are nephrolithiatic groups treated with citrate for

Chapter 8
21 and 42 days.
Data are represented as mean SD for six observations. * represents significant difference from all the control groups, c represents significant difference from
the controls, $ represents significant difference from the controls and 25 mg/ kg b.wt. astaxanthin controls, represents significant difference from the
nephrolithiatic group,represents significant difference from both the 25 mg/ kg b.wt. astaxanthin treated groups, represents significant difference from both
the 50 mg/ kg b.wt. astaxanthin treated groups, represents significant difference from both the 25 and 50 mg/ kg b.wt. astaxanthin treated for 21 days groups
and represents significant difference from all the control groups and astaxanthin treated groups. p<0.05 was considered significant
Effect of Astaxanthin on Antioxidant Status, Histology and 203

There was a significant depletion in the GSH levels in the

nephrolithiatic group (p<0.05). The treatment with 25 mg/kg b.wt.
astaxanthin did not restore the GSH levels to normal. The 50 mg/kg b.wt.
astaxanthin treated group at 42 days duration showed an elevation in the
GSH levels when compared to all the other treated groups and the values
were similar to the control groups.

Treatment with citrate at both doses increased the GSH levels when
compared to the nephrolithiatic group but the values remained significantly
lower than all the control groups and 50 mg/kg b.wt. astaxanthin treated
groups (p<0.05).
204 Chapter 8

8.1.2.5. Effect of astaxanthin on Vitamin C and Vitamin E in Kidney

Table 8.2 Vitamin C and Vitamin E in Kidney

Vitamin E
Vitamin C (g/g
(g/g wet
GROUPS wet weight of
weight of
tissue)
tissue)
Control-21 days 8.390.90 37.042.67
Control-42 days 8.390.90 37.042.67
Astaxanthin 25mg/kg b.wt.- 21 days control 9.720.25 35.230.49
Astaxanthin 25 mg/kg b.wt.- 42 days control 10.862.02 39.092.99
Astaxanthin 50 mg/kg b.wt.- 21 days control 12.862.02$ 45.783.9$
Astaxanthin 50 mg/kg b.wt.- 42 days control 14.891.44$ 48.882.76$
Nephrolithiatic 2.790.33* 9.100.65*
Nephrolithiatic treated astaxanthin 25 mg/kg
6.820.12 22.952.87*
b.wt.-21 days
Nephrolithiatic treated astaxanthin 25 mg/kg
10.660.89* 36.991.04
b.wt.-42 days
Nephrolithiatic treated astaxanthin 50 mg/kg
8.281.56 22.561.44*#
b.wt.-21 days
Nephrolithiatic treated astaxanthin 50 mg/kg
12.200.88 $# 36.381.57
b.wt.-42 days
Nephrolithiatic treated with citrate-21 days 6.220.28 22.132.98*
Nephrolithiatic treated with citrate-42 days 6.650.33 20.981.90 *

Data are represented as mean SD for six observations. * represents significant

difference from all the control groups, c represents significant difference from the
controls, $ represents significant difference from the controls and 25 mg/kg b.wt.
astaxanthin controls, represents significant difference from the 50 mg/ kg b.wt.
astaxanthin controls, represents significant difference from the 25 mg/kg b.wt.
astaxanthin treated for 21 days group, represents significant difference from the
nephrolithiatic group, represents significant difference from both the 50 mg/kg
b.wt. astaxanthin treated groups, represents significant difference from both the 25
and 50 mg/kg b.wt. astaxanthin treated for 21 days groups, represents significant
difference from both the 25 and 50mg/ kg b.wt. astaxanthin treated for 42 days
groups and represents significant difference from all the control groups and
astaxanthin treated groups. p<0.05 was considered significant.
Effect of Astaxanthin on Antioxidant Status, Histology and 205

The values of the vitamin C and vitamin E levels are represented in

Table 8.2. A significant increase was observed in the levels of the
intracellular antioxidants vitamin C and vitamin E in the astaxanthin
administered controls at 50 mg/kg b.wt. at both durations. Induction of
calcium oxalate nephrolithiasis caused a significant depletion of both the
vitamins in the renal tissue. Astaxanthin treatment significantly increased the
levels of both the vitamins from the nephrolithiatic group levels. All these
changes were significant at p<0.05. The vitamin C levels of the 50 mg/kg
b.wt. astaxanthin group at 42 days showed a higher value than other treated
groups. The vitamin E levels were comparable to controls in the 42 days
treatment at both the doses.

Treatment with citrate increased the vitamin C levels when compared

to the nephrolithiatic group but the values remained significantly lower than
all the controls and astaxanthin treated groups (p<0.05). The vitamin E levels
also increased significantly when compared to the nephrolithiatic group but
remained lower than the controls. The vitamin E levels for citrate treatment
were close to the astaxanthin treated groups at both doses for 21 days.
206 Chapter 8

8.1.3. Effect of astaxanthin treatment on the histology of the kidney

tissue - Haematoxylin and eosin (H&E) staining

8.1.3.1. Histopathology of the kidney tissue (H&E staining)

Effect of Astaxanthin on Antioxidant Status, Histology and 207
208 Chapter 8

Figure 8.5 Histopathology of the kidney tissue x400

NPL- nephrolithiatic, Asx-astaxanthin, G represents the glomerulus, represent

the proximal tubules, represent the distal tubules and the represent calcium
oxalate crystal depositions. A renal proximal tubule x1000 is labeled A.

Haematoxylin and eosin (H&E) staining of the tissues from the control
groups did not show any lesions. The glomeruli and the tubules appeared
normal. Calcium oxalate deposits composed of three to four large polygonal
crystals were abundantly found in the proximal tubules, loops of Henle, distal
tubules, collecting ducts, and even the kidney calyces in the nephrolithiatic
group. Renal tubular dilation with epithelial damage was also observed on
pathology examination. There was severe damage in the cortex as appreciated
by multifocal infarcts. Glomerular damages included mild to moderate
sclerosis of the basement membrane, widening of the Bowmans space and
atrophy of endothelial cells. Congestion of capillaries and occasional
proliferation of mesanglial cells were observed in some of the glomeruli. The
remaining structures underwent moderate to severe coagulation necrosis with
or without regeneration. The epithelium of the proximal convoluted tubules
Effect of Astaxanthin on Antioxidant Status, Histology and 209

was hypertrophic and often underwent degeneration and sloughed off in to the
lumen forming casts: cellular, granular, waxy etc. Mild to moderate
haemorrhage and occasional haemosiderosis were present. Multifocal
interstitial nephritis with moderate to severe infiltration of inflammatory cells
especially was common. Prominent vasa recta, mild interstitial fibrosis and
dilated collecting tubules stuffed with oxalate crystals were the significant
lesions in the medulla. Astaxanthin administration at 25 mg/kg b.wt. for 21
days still showed signs of tubular dilations and occasional crystal deposits
where as the 42 days treated group showed very few crystals but signs of
tubular damages were absent. The 50 mg/kg b.wt. treated group exhibited
renal histology similar to the control groups. The normal morphological and
microscopical anatomical architecture of the nephrons was retained. Certain
kidney tubules even showed regeneration of the epithelial cells. The renal
tubules of the citrate treated groups at both durations exhibited morphological
characters similar to the nephrolithiatic group with crystal depositions seen in
most tubules. There were also tubular dilations with degenerated tubular
epithelium (Figure 8.5).
210 Chapter 8

8.1.3.2 Effect of astaxanthin on renal tubular damage

Table 8.3 Renal tubular damage score

Tubular damage
GROUPS
score
Control-21 days -
Control-42 days -
Astaxanthin 25 mg/kg b.wt.- 21 days control -
Astaxanthin 25 mg/kg b.wt.- 42 days control -
Astaxanthin 50 mg/kg b.wt.- 21 days control -
Astaxanthin 50 mg/kg b.wt.- 42 days control -
Nephrolithiatic +++
Nephrolithiatic treated astaxanthin 25 mg/kg b.wt.-21 days ++
Nephrolithiatic treated astaxanthin 25 mg/kg b.wt.-42 days +
Nephrolithiatic treated astaxanthin 50mg/kg b.wt.-21 days +
Nephrolithiatic treated astaxanthin 50 mg/kg b.wt.-42 days -
Nephrolithiatic treated with citrate-21 days ++
Nephrolithiatic treated with citrate-42 days ++

- , +, ++, +++ represents 0-10%, 10-20%, 20-40% and more than 50% renal tubular injury
respectively.

The tubular damage score is given in Table 8.3. There are no tubular
damages in the renal tissue of the control animals where as the nephrolithiatic
animals show maximum number of damaged tubules. Treatment with
astaxanthin reduced the tubular damage considerably with the 25 mg/kg b.wt.
group showing moderate damage and the 50 mg/kg b.wt. group for 21 days
showing very little damage. The 50 mg/kg b.wt. group for 42 days group shows
healthy tubules similar to the controls. The tubular damage score of the citrate
treated groups at both the durations show a slight reduction in severity with 20-
40% damage.
Effect of Astaxanthin on Antioxidant Status, Histology and 211

8.1.4. Effect of astaxanthin on calcium oxalate deposition in the kidney

8.1.4.1. Pizzolato staining- Kidney
212 Chapter 8

Figure 8.6 Pizzolato staining- Kidney x100

G represents the glomerulus, represent the proximal tubules, represent the

distal tubules and the black colored masses represent calcium oxalate crystal
depositions. NPL- nephrolithiatic, Asx-astaxanthin
Effect of Astaxanthin on Antioxidant Status, Histology and 213

Pizzolato staining is done specifically for calcium oxalate crystal

identification. The calcium oxalate crystals are seen as black deposits usually
aggregated together. Crystal depositions were seen in all the six renal tissue
samples taken from the groups of the nephrolithiatic group. In the
astaxanthin treated groups, the 25 mg/kg b.wt. treated group at 21 days and
the 42 days showed crystal depositions in all the six samples which were
lesser in number than the nephrolithiatic group. The 50 mg/kg b.wt. treated
group at 21 days showed crystal deposition in three out of six samples and in
the 42 days group, no samples had crystal depositions. The citrate treated
groups at both the durations had deposition of calcium oxalate crystals in all
the tissue samples which were identical in number to that of the
nephrolithiatic group (Figure 8.6).
8. 1.4.2. Quantitative estimation of calcium oxalate crystal deposition from Pizzolato staining

214
Figure 8.7 Calcium oxalate crystal deposition in the kidney tissue
C(21D) and C(42D) are the 21 and 42 days controls, Asx-25(21D)C, Asx-25(42D)C, Asx-50(21D)C and Asx-50(42D)C are groups treated with
astaxanthin 25 mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL is the nephrolithiatic
group. NPL+ Asx-25(21D), NPL+ Asx-25(21D), NPL+ Asx-25(42D) and NPL+ Asx-50(21D) are nephrolithiatic groups treated with astaxanthin 25

Chapter 8
mg/kg b.wt. for 21 and 42 days and astaxanthin 50 mg/kg b.wt.-for 21 days and 42 days respectively. NPL+ Asx- CIT (21D) and NPL+ CIT (42D)
are nephrolithiatic groups treated with citrate for 21 and 42 days. The percentage reduction in crystal deposition is represented in parenthsis.
Data are represented as mean SD for six observations. The percentage decrease in the values from the control groups are marked in parenthesis
above the bars representing treated groups.
Effect of Astaxanthin on Antioxidant Status, Histology and 215

The number of crystals per cm2 in the cut area in 10 high power fields
was counted. There were no crystals in the control sections. The nephrolithiatic
group showed significant crystal deposition in the tubules. On astaxanthin
treatment the crystal deposition reduced with duration in the 25 mg/kg b.wt.
group by 52 % at 21 days and 91% at 42 days . The percentage of crystal
deposition was reduced by 98.62% of that in the nephrolithiatic group in the
tissue of the 50 mg/kg b.wt. group treated group at 21 days. No crystals were
seen in the renal tissue of 50 mg/kg b.wt. group at 42 days group. There were
calcium oxalate deposits in the citrate treated groups which reduced with
duration. The percentage of crystal deposition was reduced by 24 % in the 21
days groups and 42.2 % in the 42 days group (Figure 8.7).
216 Chapter 8

8.1.5. Effect of astaxanthin on calcium levels in the kidneys - Flame

photometry
Table 8.4 Calcium levels -Kidney
GROUPS Calcium (g/g)
Control-21 days 210.739.16
Control-42 days 200.739.16
Astaxanthin 25 mg/kg b.wt.- 21 days control 207.765.93
Astaxanthin 25 mg/kg b.wt.- 42 days control 199.765.93
Astaxanthin 50 mg/kg b.wt.- 21 days control 197.675.93
Astaxanthin 50 mg/kg b.wt.- 42 days control 204.765.93
Nephrolithiatic 355.367.76*
Nephrolithiatic treated astaxanthin 25 mg/kg b.wt.-21 days 238.569.95*
Nephrolithiatic treated astaxanthin 25 mg/kg b.wt.-42 days 212.65 7.77
Nephrolithiatic treated astaxanthin 50 mg/kg b.wt.-21 days 198.4511.23
Nephrolithiatic treated astaxanthin 50mg/kg b.wt.-42 days 200.0915.9
Nephrolithiatic treated with citrate-21 days 300.0933.55
Nephrolithiatic treated with citrate-42 days 286.1111.45

Data are represented as mean SD for six observations. * represents significant

difference from all the control groups, represents significant difference from the 25
mg/kg b.wt. astaxanthin treated for 21 days group, represents significant difference
from the nephrolithiatic group, and represents significant difference from all the
control groups and astaxanthin treated groups. p<0.05 was considered significant.

The calcium content of the nephrolithiatic kidney was found to be

significantly high (p< 0.05) when compared to the control groups.
Astaxanthin treatment reduced the calcium accumulation in the
nephrolithiatic group. The group administered with 25 mg/kg b.wt.
astaxanthin for 21 days showed calcium content higher than the controls but
lower than the nephrolithiatic group. All the other astaxanthin treatments
restored the calcium levels to normalcy.
Effect of Astaxanthin on Antioxidant Status, Histology and 217

The calcium content of the citrate treated groups was significantly

reduced than the nephrolithiatic group with a duration dependent decrease.
However, the calcium content of the renal tissue was higher in the citrate treated
groups than in the astaxanthin treated groups (p< 0.05) (Table 8.4).

8.1.6. SEM-EDX analysis of the renal proximal tubular cells

218 Chapter 8
Effect of Astaxanthin on Antioxidant Status, Histology and 219
220 Chapter 8
Effect of Astaxanthin on Antioxidant Status, Histology and 221
222 Chapter 8
Effect of Astaxanthin on Antioxidant Status, Histology and 223

Figure 8.8 SEM images of the renal proximal tubular cells

The SEM images of the renal proximal tubular cells x800. The
corresponding EDX analysis graph is given with each SEM image.
NPL- nephrolithiatic, Asx- astaxanthin. The calcium content as per the
microanalysis report is represented in parenthesis along with the figure
legends. point to calcium oxalate crystals in the tubular lumens.
224 Chapter 8

Numerous Plate-shaped calcium oxy monohydrate crystals were

seen in the renal tubules of the nephrolithiatic group and a few in the
astaxanthin treated groups at 25 mg/kg b.wt. The citrate treated group also
showed high crystal deposition. SEM image of the calcium oxalate crystal at
x 5000 is shown in the figure labelled A in the Nephrolithiatic.

8.2. Discussion

Reactive oxygen species (ROS) are believed to act as mediators in various

regulatory processes and signalling pathways including activation or
inactivation of regulatory biomolecules and transcriptional factors.
Uncontrolled generation of the reactive oxygen and a reduction in the
endogenous antioxidant capacity creates oxidative stress, which may lead to
inflammation and injury (Draper & Hadley, 1990). An endogenous
protective system including antioxidant enzymes like superoxide dismutase
(SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR),
Glutathione-S- transferase (GST) and antioxidants vitamin A, C, E and
reduced glutathione (GSH)) work in tandem to scavenge the reactive oxygen
species (Michiels et al, 1994). Urine supersaturated with lithogenic ions as
well as crystals formed directly induce the production of ROS in renal
tubular cells leading to oxidative damage (Thamilselvan et al, 2003). Under
oxidative stress, there is increased conversion of glyoxal to glyoxylate via
aldehyde dehydrogenase resulting in endogenous oxalate synthesis (Lange et
al, 2012). Lipid peroxidation is a complex process and the cell membranes
enriched with polyunsaturated fatty acids are more prone to lipid
peroxidation, resulting in the loss of their fluidity and permeability
(Khajuria, 1997). Oxalate, a common end product of human metabolism has
been reported to induce lipid peroxidation and tissue damage by peroxidation
of poly unsaturated fatty acids in cell membranes (Selvam & Kurian, 1987).
Effect of Astaxanthin on Antioxidant Status, Histology and 225

Hyperoxaluria and crystalluria are associated with lipid peroxidation

in renal tubular cells. Thamilselvan et al. (2003) has confirmed that
production of thiobarbituric acid reactive substances (TBARS) like MDA
occurs in the presence of enzymuria, hyperoxaluria, and crystalluria. An
elevated concentration of malondialdehyde (MDA), a breakdown product of
lipid peroxidation is one of the markers of tissue damage (Draper & Hadley,
1990). Lipid peroxidation may result from increased superoxide formation
and decreased antioxidant system in hyperoxaluric kidneys (Huang et al,
2002). Hyperoxaluria, even without crystalluria, caused increased excretion
of enzymes of renal tubular origin and cellular injury potentiates calcium
oxalate crystal formation in hyperoxaluric rats (Khan et al, 1992). This offers
additional evidence that lipid peroxidation is a major pathway in the
mechanism involved in renal tubular cell damage during oxalate induced
experimental urolithiasis. It has been suggested that free radicals and ROS
are quenched by endogenous ketocarotenoids, such as astaxanthin (Jayaraj &
Punja, 2008). Enhanced lipid peroxidation was observed in the renal tissue of
the ethylene glycol and vitamin D3 administered group as increase in the
level of TBARS. Enhancement of MDA equivalents in the aorta, in
streptozocin induced diabetic rats, was markedly inhibited by chronic
treatment with astaxanthin in vivo (Zhao et al, 2010). Astaxanthin also
reduced the MDA levels in the kidney tissue of diabetic rats at 0.05% levels
at 12 weeks supplementation (Chan et al, 2012). This was in corroboration
with our observation where a significant reduction in the TBARS level was
seen in the renal tissue of groups treated with astaxanthin 25 mg/kg b.wt. for
42 days and 50 mg/kg b.wt. at both the durations.

Cells are endowed with several enzymatic antioxidants like SOD,

Catalase, GPx and GR and non enzymatic ones such as GSH, vitamins A, C and
226 Chapter 8

E. Cellular defences are unable to withstand oxidative insult once their threshold
is exceeded. Oxalate has been documented to cause renal tubular injury by
increasing generation of free radicals (Scheid et al, 1996; Bhandari et al, 2002).
Oxalate induced lipid peroxidation in renal tubular epithelial cells in culture was
associated with a greater production of free radicals. This was found to be
greater when the cells are exposed to oxalate and calcium oxalate monohydrate
crystals. This points out that oxalate itself is injurious to cells and that calcium
oxalate crystals potentiate the toxicity (Thamilselvan et al, 2000). Exposure to
high concentrations of oxalate can induce oxidative stress as shown by increased
lipid peroxidation (Thamilselvan et al, 1997) decreased glutathione
concentrations (Muthukumar & Selvam, 1998) and decrease in the activity of
antioxidants (Meimaridou et al, 2006). The decrease of the GPx enzyme
determines the accumulation of ROS and the oxidation of membrane lipids. The
regeneration of GSH is mediated by GR on the expense of NADPH, thus the
decrease of GR may also leads to increase in oxidative stress.

A previous study by Huang et al. (2002) on antioxidant enzyme

levels in rats with stone formation showed that almost all the antioxidant
enzyme activities were attenuated. Our results are also in confirmation with
this observation where depletion in the level of the antioxidant enzymes
SOD, Catalase, GPx, GR and GST was seen in the renal tissue of the
nephrolithiatic group.

SOD catalyzes superoxide anion radical dismutation into hydrogen

peroxide. It is logical to believe that low SOD levels represent a
generalised deficiency of this enzyme in the tissues leading to increased
flux of superoxide anion resulting in increase in oxygen related free
radicals and thus greater peroxidation which shall result in higher levels of
peroxidative products like TBARS in tissues and blood. SOD inhibition
Effect of Astaxanthin on Antioxidant Status, Histology and 227

might be also a consequence of an excess of reactive oxygen species, which

would affect enzyme structure (Salo et al, 1988). Previous reports
demonstrated that astaxanthin protects against ethanol and naproxen-
induced gastric lesions by its ability to restore the activities of SOD, GPx
and CAT (Kim et al, 2005). A similar study on astaxanthin isolated from
H. pluvialis was conducted and identical results were obtained (Kamath et
al, 2008). Additionally, astaxanthin reduced the oxidative stress in kidneys
and prevented renal cell damage caused by diabetes in mice (Naito et al,
2004). All these studies support our results where astaxanthin
administration enhanced and restored the activities of the enzymes SOD,
catalase, GPx, GR and GST. Astaxanthin supplementation significantly
increased the expression of Nrf2 and most of its target genes in the livers of
apoE2/2 mice (Yang et al, 2011). Nrf2 pathway responsive genes include
glutathione synthetase (GS), glutathione-S-transferase (GST), GPx, and
SOD (Aleksunes et al, 2009). The increased expression of its downstream
targets may provide a stronger endogenous defensive system against
oxidative stress in the body. A stimulatory effect was seen on GPx and GR
activity in healthy rats by astaxanthin administration in studies by Leite et
al. (2010). Such an induction in the renal GR and GST levels were
observed in our study also in the astaxanthin controls fed with
50 mg/kg b.wt. astaxanthin at both durations.

The imbalance in the glutathione system may cause of a change in

the level of GSH, a compound with important biological functions
including cell differentiation, proliferation, apoptosis, immune system
activation and action in several human diseases (Ballatori et al. 2009).
Depletion in the tissue antioxidant GSH is noted in the nephrolithiatic
animals. This may be due to depressed activity of NADPH-dependent GR
228 Chapter 8

required for the conversion of GSSG to GSH. A concomitant decline in the

levels of the vitamins C and E is noted in stone induced animals.
Thamilselvan & Selvam (1997) has reported that the renal antioxidants
vitamin E, ascorbic acid and glutathione were significantly decreased on
oxalate challenge. Previous reports by Selvam & Kurien (1992) showed
that in calculi producing diet fed rats, the activities of antioxidant enzymes
decreased along with a diminished vitamin E, ascorbic acid and tissue
glutathione content. However, the GSH and the vitamin C and E levels
were drastically enhanced on treatment with astaxanthin at all doses and
duration.

The electron transfer from isoflavonoids to the carotenoid radical

cation formed during oxidative stress is faster for astaxanthin than for the
other carotenoids which may relate to more effective antioxidative
properties of astaxanthin and is in agreement with its highest electron
accepting index (Han et al, 2010). The 13 conjugated double bonds and the
hydroxyl groups in the 3 and 3 positions make the molecule highly polar
and dramatically enhance its membrane function to protect against
degenerative conditions. This is in contrast to other carotenoids, which
gives it significantly greater antioxidant capacity (Shibata et al, 2001).
Because of its polar end groups, astaxanthin spans the cell membrane
bilayer allowing it to sit near the lipid/water interface, where free radical
attack first occurs and contributes to cell membrane mechanical strength
(Palozza and Krinsky, 1992). ). It can resist chain reactions that occur when
a fatty acid is oxidized. It does so by stabilizing free radicals by adding
them to its long double-bond chain rather than donating an atom or electron
to the radical (Kurashige et al, 1990).
Effect of Astaxanthin on Antioxidant Status, Histology and 229

The administration of astaxanthin is also found to decrease the

calcium accumulation in the renal tissue. Flame photometry and Pizzolato
staining too support this observation as the number of calcium oxalate
deposits decreased in the treated rats. Light microscopic examination of the
tissue revealed polycrystalline rosette shaped crystals in the tubular lumens
of the ethylene glycol treated rats which is an obvious evidence of adhesion
and retention of calcium oxy monohydrate crystals. Defects in oxalate
metabolism produces tubulointerstitial lesions which is recognized as one
of the most important risk factors for the development of chronic renal
diseases and eventual renal failure (Nath, 1998).The crystal containing
regions of the tubules showed dilations and degeneration of their lining
epithelia. The histological analysis showed an improved renal tissue in the
treated group with fewer crystals in the lumen of renal tubules and lesser
extent and number of tubular dilations. This suggests the ability of
astaxanthin to act as a calcium oxalate crystal aggregator inhibitor
preventing the development of calcium oxalate calculi in addition to its
antioxidant effect. A previous report has shown that membrane damage can
be reduced by supplementation of antioxidants in various pathological
conditions (Buoncristiani et al, 1997). Antioxidants like vitamin E,
glutathione monoester or methionine or lipoic acid normalized the cellular
antioxidant system and prevented precipitation of salts in the rat kidney
(Selvam, 2002) and reduced oxalate excretion in stone patients
(Anbazhagan, 1999). Astaxanthin has been demonstrated to protect in vitro
against oxidation of lipids at the surface and inside phospholipid
membrane. This effectiveness can be explained by location of astaxanthin
inside in membranes, where the polar ring of astaxanthin would scavenge
reactive oxygen species near the membrane surface, while the polyene
230 Chapter 8

chain would inhibit the radical chain reaction into the membrane (Goto et
al, 2001). Thus astaxanthin can effectively reduce lipid peroxidation,
enhance the activities of enzymatic and non enzymatic antioxidants in
experimentally induced calcium oxalate nephrolithiasis. Treatment with
astaxanthin also reduces calcium oxalate crystal deposition and restores the
integrity of the renal tissue. This could possibly be due to its antioxidant
effect and its ability to influence glomerular filtration rates.