Antioxidant Effect of Clorella vulgaris on Wistar Rat Kidney Induced by CCl

Kidney is very susceptible to damage by toxicological compounds such as carbon tetrachloride (CCL). CCL produce free radicals, which cause lipid peroxidation and kidney damage and free radical release, which can be prevented by the administration of exogenous antioxidants, such as Chlorella vulgaris. The aim of study was to determine an effect of antioxidant of C. vulgaris on the histopathological features of Wistar rat kidney which is induced by CCL. Experimental study with completely randomized design. The variable was histopathology features of the kidneys. The doses of C. vulgaris extract were 3 mg, 4 mg, and 5 mg per 100 grams of rat body weight (BW). The administration of C. vulgaris extract was performed within 30 days, while the CCl 4 induction (0.25 ml/100 g BW) was administered orally on the day 9, 12, 16, 19, 23, and 26. Parameters were histopathology features of renal damage, proportion of tubular cell damage, and Bowman’s space diameter. The results showed the administration of C. vulgaris extract was able to reduce the impact of damage caused by CCl 4 (p<0.05). This was supported by histologic observations, which was showing a decrease of picnotic and vacuolated cells, normal brush border, and decrease of Bowman’s space. In conclusion, 5 mg/100 g BW of C. vulgaris extract can effectively protect the kidney from damage caused by CCL. The results of this study strongly support further research on immunostimulant content test C. vulgaris and determine the efficient dose for representative mammals animals also in humans.

wildering array of opportunities to develop healthier food products using innovative approaches and a number of different strategies.Compared to other natural sources of bioactive ingredients, microalgae have many advantages such as their huge biodiversity, the possibility to grow in arid land and with limited fresh water consumption and the flexibility of their metabolism, which could be adapted to produce specific molecules.All these factors led to very sustainable production making microalgae eligible as one of the most promising foods for the future, particularly as source of proteins, lipids and phytochemicals (Buono et al., 2014).
C. vulgaris is one of many food, which contains abundant amount of antioxidant.According to a study by Cha et al. (2010), there were 15 type of antioxidants within C. vulgaris extract.These antioxidant are including in lipophilic and hydrophilic compounds, which is capable of reducing oxidative effect of free radicals (Satish & Dilipkumar, 2015).
Study about potential of C. vulgaris as antioxidant to prevent kidney damage was never been performed.Therefore, this study aimed to understand the effect of C. vulgaris extract in reducing kidney damage, which was exposed to CCl 4 .We studied the effect of C. vulgaris extract administration on histopathological features of wistar rat (Rattus norvegicus) kidney and its dose that effective in reducing kidney damage in Rattus norvegicus which was induced by CCl 4. Result of this study will advance the knowledge about antioxidant potential of C. vulgaris and open the possibilities of further study in human body.

Animal Test and Study Ethic
Animal used in this study were 30 male white rats (Rattus norvegicus) wistar strain with 2-3 month of age and 100-150 g of weight from UD Wistar, Yogyakarta.Permission for using these animal has been granted by Ethical Committee of Health Research, Dr. Moewardi General Hospital, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, number 680/VII/HREC/ 2017.

Tools and Materials
Tools used in this study were cage (40x30x15 cm), drinking bottle, gastric probe, and a set of equipment for tissue processing and a microscope for histologic observation.
Materials used in this study were 30 male Rattus norvegicus wistar strain with 2-3 month of

INTRODUCTION
Kidney has an important role in filtering metabolic waste products and toxic compounds on blood.This role involves tubular structure and counter-current on medulla, which play role in 25% of circulated blood reabsorption within the body (Weiner et al., 2015).The high percentage of filtered blood makes this organ vulnerable toward damage due to toxic compound (nephrotoxic) within the blood.
Generally, nephrotoxic compound causes damage on kidney and reduces its function, such as excretion of metabolic waste products, synthesis of erythropoietin, maintain electrolyte balance, and maintain fluid balance (David et al., 2012).
One of the nephrotoxic compound is carbon tetrachloride (CCl 4 ).Its toxicity is come from free radical from its metabolism by cytochrome P450 enzyme.Free radicals are atoms or molecules that have one or more unpaired electrons.This causes them are chemically reactive, which can cause chemical changes and damage on various components of living cells.In the human body, free radicals are considered to play a role in the occurrence of some diseases, such as aging (Fitmawati et al., 2017).This compound will be metabolized into trichloromethyl (CCl 3 -) (Barsha et al., 2017).Trichloromethyl reacts to oxygen molecule and forms trichloromethyl peroxy (CCl 3 COO -), which is a free radical (Fouw, 2007).This radical causes lipid peroxidation of phospholipids in cellular membrane and kidney cells damage (Antonio et al, 2014).
Impact of damage caused by free radicals can be reduced using antioxidant as protective agents.These agents are produced within the body continuously in the form of endogenous antioxidant (Hsu et al., 2013).However, increased free radicals within the body can reduce effectivity of endogenous antioxidants as protective agents.Therefore, exogenous antioxidant is needed.Several example of exogenous antioxidants are phenolic and carotenoid compound within microalgae Clorella vulgaris (Goiris, 2012).
A wide variety of natural sources are under investigation to evaluate their possible use for new functional ingredient formulation.Some records attested the traditional and ancient use of wild harvested microalgae as human food but their cultivation for different purposes started about 40 years ago.The most popular species are Arthrospira (traditional name, Spirulina), Chlorella spp., Dunaliella spp.and Haematococcus spp (Tang & Suter, 2011).Microalgae provide a be-age and 100-150 g of weight; C. vulgaris extract with 3 mg, 4 mg, and 5 mg of doses; rats feed, husk, CCl 4 solution, physiologic NaCl, olive oil, and a package of chemical reagents for tissue processing.

Location and Time of Study
This study was performed within 6 month in Animal House, Laboratory of Animal Physiology, and Laboratory of Animal Structure and Development of Faculty of Biology Jenderal Soedirman University.We also conducted the experiment in Laboratory of Pharmacy, Jenderal Soedirman University.

Production of C. vulgaris Extract
Production of ethanol extract was performed using maceration method (Cha et al., 2010).Dry C. vulgaris weighed as much as 50 g and added to 100 ml of 96% ethanol in beaker glass.This solution was mixed and left for 24 hour until it was sedimented.After 24 hour, the maserate was taken and placed in clean beaker glass.Furthermore, the maserate was resolved by using 96% of ethanol (remaseration).Remaseration was performed three times in 24 hour, repeatedly until the color of the mixture faded.
Obtained solution was evaporated using Vacuum Rotary Evaporator to remove solvent and to obtain thick C. vulgaris extract.

Experimental Design
This study used experimental method with complete random design, which was consisted of 6 treatment and 4 repetition.The 6 treatment were consisted of K+, KS, K-, P1, P2, and P3.Before the experiment performed, the rats in each treatment was acclimated within 10 day with pellet feed twice a day (on 7 AM and 4 PM).Furthermore, the feeding was continued routinely until 30 days of experiment with the same time of administration.
For 30 days of experiment, the rats in KS, P1, P2, and P3 treatment groups were given C. vulgaris extract twice a day (before meals) with dose 2 mg/ 100 g BW, 3 mg/ 100 g BW, 4 mg/ 100 g BW, and 5 mg/ 100 g BW, respectively, using gastric probe.Induction using 0.25 ml/100 g BW of CCl 4 was administered to K-, P1, P2, and P3 treatment groups using gastric probe on day 9, 12, 16, 19, 23, and 26.Physiologic NaCl were given to rats in K+ and KS group, which were free of CCl 4 induction.

Histologic Preparation
After 30 days of treatment, on the day 31, wistar rats were decapitated using ether.Furthermore, these rats were dissected and their left kidney were extracted.The kidney was dissected and cut using surgical tools.Left kidney was isolated, washed using physiologic NaCl, and fixated in neutral buffered formalin solution for 48 hour.After 48 hour, the kidney was processed into histologic preparation using paraffin method and was stained using Haematoxylin-eosin stain (Arsad et al., 2014).

Histologic Evaluation
Histologic preparation of kidney was observed using light microscope.We observed the proximal tubule in cortex and the diameter of Bowman's space in 5 field of each slice using 400 fold of magnification.Measurement of Bowman's space diameter was performed by calculated the difference between Bowman's capsule and glomerulus (Kotyk et al., 2015).Calculation of damaged proximal tubule cell was performed using Modified Cell Degeneration Index Formula (Maulina, 2015) as follows: Cell Degeneration Index of Tubule = (Amount of Cell Necrosis /Amount of Total Cell) x 100%

Data Analysis
Data of this study were consisted of cell degeneration index of kidney tubules data, Bowman's space diameters data, and histopathological image of kidney.Qualitative data of kidney histology were analyzed descriptively.Cell degeneration index data were transformed into arc.Sin .Furthermore, kidney weight, cell degeneration index of kidney tubules, and Bowman's space diameters were analyzed using ANOVA with significant level of 5% (p=0.05).If the result show significant value, it will be analyzed with post hoc test using HSD Tukey.

RESULTS AND DISCUSSION
According to the obtained result (Table 1), the tubule of rats on K-treatment group, which was inducted by CCl 4 without C. vulgaris extract administration, were damaged with high average value of 47.84% ± 0.04.This value was far greater than the average value of K+ treatment group, which was neither inducted by CCl 4 nor received C. vulgaris extract, with damage percentage of 9.96% ± 0.02.The amount of cell degeneration index of tubules in K-treatment group showed significant damage on proximal tubule due to CCl 4 .The damage was happened due to accumulation of nephrotoxic compound and its biotransformation by P450 cytochrome (Tugba et al., 2014).This result was showed damage on tubule cell of wistar rats after administration of 0.25 ml/100 g BW CCl 4 .The study showed that the nucleus became picnotic after CCl 4 administration.Current study also showed dilatation, vacuolation, and brush border loss of tubule cell.These findings also relevant to study by Ogeturk (2005), Adewole (2007), and Ashafar (2008), which showed similar damage on tubule cell of wistar rats after CCl 4 administration.
Percentage of tubule cell damage on P1, P2, P3, and KS treatment group were 23.17% ± 0.04; 20.59 ± 0.007; 14.60% ± 0.02; and 9.58% ± 0.01, respectively.Percentage of tubule cell damage on P1, P2, and P3 treatment group were less than percentage of tubule cell damage on K-treatment group.These findings proved the ability of C. vulgaris extract in preventing cell damage due to CCl 4 exposure.These findings were also supported by normal histologic features of the cell with no vacuolation, no dilatation, few picnotic nucleus, and normal brush border.
Result of statistical analysis using ANOVA showed that administration of C. vulgaris extract produced significant effect on cell degeneration index of tubule cell (p<0.05).Further test using HSD Tukey (Table 2) showed similar notation value between P1 and P2 (p>0.05).Similar notation value was also shown on HSD Tukey test between P2 and P3 (p>0.05).On the other words, small range of C. vulgaris extract dose on P1, P2, and P3 treatment group did not produced significant difference.
Notation value of P3 treatment group also showed similar result with K+ and KS treatment group, which means the administration of 5 mg/100 g BW C. vulgaris extract was not capable to reduce the percentage of tubule cell damage.However, administration of C. vulgaris extract was capable to produce sufficient protection against oxidative stress, which was proved by significant difference of notation value between K-and other treatment group (P1, P2, and P3).
Result of measurement of Bowman's Space diameter showed that wistar rats on Ktreatment group had the biggest average (7.04 µm ± 1.71) compared to other treatment groups (Table 1).Average value of K-treatment group was far above average value of K+ treatment group (3.50 µm ± 1.09).Dilatation of Bowman's space and shrinkage of diameter (atrophy) of glomerulus, which is shown in Table 1, indicated degeneration and disintegration of glomerulus (Ogeturk, 2005) and Hismiogullari et al. (2015), which showed dilatation of Bowman's space and atrophy of glomerulus on wistar rat's kidney after administration of CCl 4 .The diameter of Bowman's space on P1, P2, and P3 treatment groups were 5.24 µm±0.86,4.21 µm±1.63, and 4.00 µm±1.07,respectively.The three of these treatment groups had smaller Bowman's space diameter than K treatment group.It means the administration of C. vulgaris extract as antioxidant is capable of protecting kidney from damage.
Statistical analysis using ANOVA proved that administration of C. vulgaris extract produce significant effect on Bowman's space diameter with p<0.05.Further analysis using HSD Tukey (Table 2) showed administration of 3 mg/100 g According to the result of observation and calculation, we found that the increase of tubule cell damage percentage was reciprocal to Bowman's space diameter.Dilatation of Bowman's space was followed by decrease of glomerular diameter.However, this study was not capable to provide the actual reason, which could explained the relationship between these two parameters, using histological observation only.Therefore, we strongly recommended to perform further study about urea, creatinine, and renal malondialdehyde (MDA) on serum, which are generally used to assess renal function (Ogeturk, 2005).
The reason behind the relationship between tubule cell damage and Bowman's space has been reported on several studies.The tubule cell damage interrupts reabsorption process and slows urinary flow from renal corpuscle to tubule cell.Furthermore, renal corpuscle size is increasing due to urinary accumulation on Bowman's space.Another study reported that Bowman's space dilatation was caused by increase of Glomerular Filtration Rate (GFR) due to sodium reabsorption process and hyper filtration on glomerulus.In result, tubuloglomerular feedback will be reduced and Bowman's space will be dilated (Helal, 2012;and Henegar, 2001).
Result of histologic observation on P1, P2, and P3 treatment group showed that C. vulgaris extract was capable of protecting kidney from oxidative stress after administration of CCl 4 , which is a nephrotoxic compound.These result was shown by tubule cell degeneration index and Bowman's space diameter.Observation result also proved that an increase of C. vulgaris extract dose was followed by a decrease of kidney damage due to CCl 4 .This statement was supported by the fact that P3 treatment group had the smallest tubule cell degeneration index and Bowman's space diameter.This protective nature of C. vulgaris extract was reciprocal to a study by Blas-Valdivia et al. (2011), which proved that administration of C. vulgaris extract capable of reducing cell damage and cortical atrophy due to HgCl 2.Not only protecting kidney from nephrotoxic compounds, C. vulgaris also capable of protecting kidney damage due to diabetes mellitus (Aizzat, 2010).The ability of C. vulgaris extract to provide protection against oxidative stress and free radical is related to antioxidant compound inside it.Several antioxidants in this extract are lutein, acarotene, b-carotene, ascorbic acid, chlorophyll, and tocopherol (Cha et al., 2010).According to a study by Blas-Vladivia (2011), the most important antioxidant, which had a role in preventing damage due to free radicals, is carotene.This compound can remove single oxygen and peroxide compounds through redox reaction (Jian-Ming et al., 2010).The results of this study strongly support further research on immunostimulant content test C. vulgaris and determine the efficient dose for representative animals mammals also in humans.

CONCLUSION
According to result and discussion, can be concluded that the administration of C. vulgaris extract can protect white wistar rat (Rattus norvegicus) kidney from damage, which were picnotic cell vacuolation, brush border damage, and Bowman's space broadening due to CCl 4 exposure.The most effective dose of C. vulgaris extract, which gave protection for kidney was 5 mg/100g BW.

Figure 1 .
Figure 1.Micrograph Picture of Wistar Rat's Kidney after Administration of C. vulgaris Extract and Inducted by CCl 4 on 400 x magnification (Haematoxylin-Eosin Stain).Figure A from K+ and B from KS treatment group showed normal Brush Border (BB), glomerulus (G), Bowman's space (BS).Figure C showed picnotic of nucleus (P), vacuolation (V), diminished brush border, and dilatation of Bowman's space on K-treatment group.Treatment group of P1 (D), P2 (E), and P3 (F) showed normal condition.
Figure 1.Micrograph Picture of Wistar Rat's Kidney after Administration of C. vulgaris Extract and Inducted by CCl 4 on 400 x magnification (Haematoxylin-Eosin Stain).Figure A from K+ and B from KS treatment group showed normal Brush Border (BB), glomerulus (G), Bowman's space (BS).Figure C showed picnotic of nucleus (P), vacuolation (V), diminished brush border, and dilatation of Bowman's space on K-treatment group.Treatment group of P1 (D), P2 (E), and P3 (F) showed normal condition.
Figure 1.Micrograph Picture of Wistar Rat's Kidney after Administration of C. vulgaris Extract and Inducted by CCl 4 on 400 x magnification (Haematoxylin-Eosin Stain).Figure A from K+ and B from KS treatment group showed normal Brush Border (BB), glomerulus (G), Bowman's space (BS).Figure C showed picnotic of nucleus (P), vacuolation (V), diminished brush border, and dilatation of Bowman's space on K-treatment group.Treatment group of P1 (D), P2 (E), and P3 (F) showed normal condition.

Table 2 .
Notation Value of Tukey Test on Cell Degeneration Index of Tubule Cell and Bowman's Space

Table 1 .
Result of Histologic Examination of Wistar Rats Kidney