Introduction
The beneficial effects of olive oil are mainly due to its high oleic acid content. Its strong antioxidant properties are due to the presence of phenolic components. The kidney performs its vital role in the infiltration of blood and excretion of waste compounds through the various parts of its nephron.[1] It has various parts which including the Bowman’s capsule which surrounds the glomerulus. Filtration of blood takes place in the medulla through a loop of Henle and collecting tubules, both of which are found in the medulla.[2] Among various metalloids which affect the organs of the human body, arsenic is a naturally occurring metalloid. It is the most predominant global environmental toxin which causes oxidative damage to the kidney. Arsenic in drinking water causes proteinuria, albuminuria, and chronic kidney disease (CKD) which is determined by decreased glomerular filtration rate.[3] The renal biopsies of patients who are exposed to arsenic showed acute tubular necrosis causing acute renal failure. Some patients may develop renal cortical necrosis (RCN), diffuse interstitial fibrosis which ultimately leads to CKD.[4] Arsenic in drinking water is about 200–300 μg per l. This causes a high mortality rate due to diabetes mellitus, kidney disease, and cardiovascular diseases. The inorganic arsenic is distributed throughout the body and is taken up by the cells in tissues.[5]
It is present in water, soil, and even in some types of food.[4] As a result of arsenic contamination from industrial operations and groundwater overdrawn for irrigation, drinking water is the primary source of arsenic exposure to living organisms (0.01–3.7 mg/l).[6] Arsenic contaminated groundwater is extensively utilized both for irrigation and drinking nearly in all areas of Pakistan. The groundwater of 32 districts of Punjab is contaminated with a toxic level of arsenic. According to the World Health Organization (WHO), drinking water should be having a cutoff value for arsenic to be <10 μg/l.[7] A nationwide survey of drinking water conducted in 35 of 104 districts show that only 9% of samples have arsenic levels meeting the WHO standard of 10 ppb. In Sindh, the arsenic level in groundwater is 96 mg per l and 157 mg per l in surface water.[8]
In Pakistan, an important cause of CKD is the occurrence of arsenic and heavy metals in the feed of poultry chicken. Therefore, arsenic can be transmitted to the human body by eating chicken.[1,9] The increased production of reactive oxygen species (ROS) by arsenic causes raised lipid peroxidation in the hepatic and renal tissue. The activity of antioxidant enzymes such as superoxide dismutase and catalase is decreased leading to reduced glutathione content in the kidney.[10,11] Distortion of cortical cellular architecture, tubular atrophy, and necrosis along with densely packed red blood cells in the glomerulus as well as both degenerative and atrophic changes have been reported. The RCN involves both tubules and interstitium.[12]
Antioxidants such as olive oil may reduce the effect of arsenic on the kidney.[3] Olive oil possesses great health benefits such as the prevention of coronary heart disease, cancers, and modification in immune responses. It has anti-apoptotic, anti-inflammatory, and anti-oxidative properties as it protects the tissues against damage caused by oxidative stress.[13] There is the ability of extra virgin olive oil (EVOO) to alter the cell membrane structure and to reduce oxidative injury of compounds.[14]
The antioxidant effect of EVOO depends upon the amount of oleuropein and tyrosol.[15] The use of olive oil has also improved renal histoarchitecture including glomerular fragmentation, enlargement of Bowman’s space, hemorrhage, infiltration of leukocytes, and tubular dilation caused by acrylamide[16] based on its properties, olive oil may be effective in protecting kidneys from arsenic-induced histological changes. In Pakistan, epidemiological data show that CKD is progressively increasing due to arsenic use[17] while its awareness is very poor among common as well as medical officers and pg trainees.[18]
The objective of our study is to determine the association of high levels of arsenic in the body with kidney damage and also find out the ameliorative effect of EVOO.
Material and Methods
The study was conducted at the Islamic International Medical College after approval from the ethical review committee. The duration of the study was 4 weeks from November 1, 2017 to November 30, 2017. Forty-five adult male albino rats, 250–300 g, were procured. All rats included were 2–4 months of age and those with any obvious congenital anomaly and female rats were excluded to avoid their mating with males. Under the supervision of animal house National Institute of Health, rats were kept in a well-ventilated room with alternating 12 h light and dark cycle at 20°C–26°C temperature. They were kept in three cages 15 rats per cage in a controlled standard living environment suitable to their class with an adjusted diet. Rats in the control group consumed distilled water as drinking water for 4 weeks. Group II rats consumed sodium arsenite (40 mg/kg) for 4 weeks, while rats in Group III sodium arsenite (40 mg/kg) along with olive oil 0.2 ml/day for 4 weeks. After 4 weeks, all rats were euthanized and their kidneys were dissected. H and E staining of control and experimental groups was done. Renal corpuscle, tubule, and interstitium were observed in the renal cortex of each slide at four different fields and the mean of all readings was taken as a single reading. The diameter of renal glomerulus and width of Bowman’s space was measured by method,[19] as shown in Figure 1.
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Figure 1: Shows the method used for the determination of glomerular radius and width of Bowman capsule with the help of triangle drawn
The following histological parameters were observed in the slides in detail under Ă—10 and Ă—40 power of light microscope by using image J software. n kidneys of the experimental group.
The diameter of proximal and distal convoluted tubule (DCT) was noted in renal tubule, whereas width of Bowman’s space and the diameter of the glomerulus were noted in the renal corpuscle.
After accomplishment of 4 weeks duration of the experiment, rats were anesthetized with chloroform and dissected.
After fixation and embedding, eosin and hematoxylin stains were used for the histological section.
Results
The leukocyte or white blood cell which includes plasma cells, lymphocytes, neutrophils, and macrophages was counted in four areas of one slide. No leukocytes are found in control Group I, whereas the count of leukocytes in Group II and Group III was 32 and 13, respectively. As P value in the comparison of Groups I and III are 0.375 which is ≥0.05, so it is insignificant. The mean width of Bowman’s space was 21.84 um in Group II, which was increased due to the use of arsenic as compared to control Group I. The use of olive oil in Group III has significantly prevented the increase in width of Bowman’s space as it was 12.55 um. As the P value in comparison of Groups I and III are 0.330 which is ≥0.05, this shows that difference of mean width of Bowman space between these two groups is insignificant showing that olive oil has significantly decreased leukocyte count. It is shown in [Table 1 and Figure 2].
Table 1: Group wise distribution and multiple comparisons of mean leukocyte count in the renal interstitium of control and experimental groups of albino rats
Figure 2: (a) Shows the change in the urinary space (μm) indicated by arrows and (b) shows the change in diameter of PCT and DCT indicated by stars Figure shows (c) olive oil group with decreased bowman’s space (d) control group, (e) arsenic group and (f) olive oil and arsenic group. Blue stars for the lumen of PCT while black stars for the lumen of DCT (a = control group, b = Arsenic group, c = Arsenic with olive oil group). PCT: Proximal convoluted tubule. DCT: Distal convoluted tubule
The mean diameter of the glomerulus in Group I was 104.72 um which was decreased to 80.86um in Group II due to the use of arsenic as Bowman’s space was enlarged. The use of olive oil in Group III prevented the decrease in glomerular diameter which was found to be 93.5 um. The P value for the comparison of Groups II and III was found to be 0.23 (≥0.05); therefore, it is statistically insignificant. This proves that olive oil has not significantly improved the decrease in the diameter of glomeruli caused by arsenic in Group III [Table 2 and Figure 2].
Table 2: Group wise distribution and multiple comparisons of a width of Bowman’s space and diameter of the glomerulus in the renal interstitium of control and experimental groups of albino rats
The mean diameter of proximal convoluted tubule (PCT) in Group I was 30.94um which was increased to 49.11um in Group II due to the use of arsenic which caused loss of brush border. The use of olive oil in Group III prevented the increase in PCT diameter which was found to be 35.95um. The P value for the comparison of all groups is <0.05 which is highly significant. This proves that olive oil has significantly prevented the increase in diameter of PCT caused by arsenic in Group III [Table 3 and Figure 2].
Table 3: Group wise distribution and multiple comparisons of the diameter of proximal convoluted tubule and distal convoluted tubule in the renal cortex of control and experimental groups pf albino rats
The mean diameter of DCT in Group I was 52.19 mm which was increased to 96.16um in group II due to the use of arsenic. The use of olive oil in Group III prevented the increase in DCT diameter which was found to be 66.04 ÎĽm. The P value for the comparison of all groups is 0.000 which is highly significant. This proves that olive oil has significantly improved the increase in diameter of DCT caused by arsenic in Group III [Table 3 and Figure 2].
All the rats in the control group showed a brush border of PCT. 87% of experimental animals in Group II showed loss of brush border of PCT. In Group III, only 5% of rats showed loss of brush border in PCT, whereas 10% showed the presence of brush border in PCT. In this way, olive oil has significantly decreased the number of rats in Group III showing loss of brush border of PCT. P = 0.000 as shown in Figure 3.
Figure 3: Shows the percentage of rats showing the presence and absence of loss of brush border
Discussion
The arsenic concentration in drinking water of various regions of Pakistan including KPK, Rahimyarkhan, and Bahawalpur is 1.56 μg/l. 10 to 50 μg/l, and 0–10 μg/l.[20] In district Multan, DG Khan, Jhang, Leiah, Mianwali, and Muzaffargarh, it is reported between 10 and 50 μg/l.[21] Among natural antioxidants, owing to a protective effect against heavy metals-induced toxicity especially when ROS are involved, olive oil is an integral ingredient. Virgin olive oil (VOO) is a good source of phytochemicals including polyphenolic compounds which have a protective effect on the kidney.[22]
In our study, leukocytes were counted in the renal parenchyma. The mean leukocyte count in Group II was found to be 32, whereas in Group III, it was reduced to 13. The leukocytes are infiltrated in the regions of RCN. RCN is either diffuse cortical necrosis or patchy cortical necrosis depending upon the area of the cortex involved. A study conducted by Alforkan from China in 2019 showed an increase in the number of inflammatory cells in the interstitium of the renal cortex of sodium arsenite-treated rats. Sodium arsenite was given in distilled water in different doses of 10, 30, and 50 ppm for 90 days.[23] In a study conducted by Hewa in 2016, mice were treated with hard water containing arsenic above 0.01 mg for 28 weeks. This long study showed CKD with increased mononuclear infiltrate in the kidney as compared to groups that were given hard water with 0.01 mg per l of arsenic. This range is acceptable according to WHO.[19]
In another study by Kharroubi et al.,[27] conducted in 2014 in Berlin, sodium arsenite of 1 and 10 mg per liter was given for 45 and 90 days and showed lymphocytes infiltration which was increased with increased duration of the study. In a study conducted by Ghorbel et al.,[16] the use of EVOO of 300 ul for 21 days has significantly reduced leukocyte count in renal interstitium caused by 40 mg per kg of arsenic.[24] Said Elshama in 2016 proved that the use of 100 mg per kg per day naringin and 1.25 ml per kg per day olive oil have renoprotective properties. Both caused a significant decrease in inflammatory cell infiltrates in rats which were treated with 25 mg per kg of cyclosporine.[16] Similarly, in the study conducted by Eman Taha Mohammad from Egypt in 2014, 5 mg/kg of CdCl2 was used orally for 4 weeks. 0.5 ml per kg of VOO markedly decreased the inflammatory cells infiltrate in rats.[25]
In a recent study, an increase in Bowman’s space diameter was caused by 40 mg per l of Arsenic for 4 weeks. In comparison to this study, Kharroubi et al. in 2014 used sodium arsenite for 45 and 90 days in a dose of 10 mg per l, this showed expansion in space between glomeruli and Bowman’s capsule.[26] In contrast to this study, another study conducted by Kumar et al.,[28] of India in June 2016, mice were administered with only 6 mg per kg of Arsenic for 4 weeks followed by lactobacillus for 8 weeks. The Bowman’s space was diminished. This was due to the dilation of glomeruli in the early stage of inflammation.[27] In one of the other studies conducted by Zongyuan of China in which arsenic was given in the low dose of 4 mg per kg for 60 days in drinking water, it caused hyperemia and dilation of glomeruli resulting in diminished Bowman’s space.[28]
Jemai et al. conducted a study in May 2015 in which treatment with olive leaf extracts markedly improved collapsed glomerular tuft causing Bowman’s space diameter almost the same as that of control rats.[31] Similarly, a study by Eman taha from Egypt in 2014 used 0.5 ml per kg of VOO orally which significantly reduced dilation of Bowman’s space caused by an oral dose of 5 mg per kg of CdCl2 for 4 weeks in rats.[29] Said Elshama in 2016 proved that use of 100 mg per kg per day naringin and 1.25 ml per kg per day olive oil significantly decreased dilation of Bowman’s space caused by 25 mg per kg of cyclosporine.[26]
In our study, the use of 40 mg per kg of arsenic increased the diameter of PCT due to loss of brush border and also increased the diameter of DCT along with degeneration of epithelium. A similar study conducted by Kharroubi et al. in 2014 in which 10 mg per l of sodium arsenite was given for 45 and 90 days produced similar results of an increase in diameter of PCT and DCT.[27] In contrast to this, a study conducted by Zongyuan et al. of China in which mice were exposed to only 4 mg per l of arsenic in drinking water for 60 days, arsenic caused swelling of epithelial cells of PCT and DCT which caused a decrease in diameter. This is the early stage of inflammation. If a toxic level of arsenic is used as in our study, it caused degeneration of cells of DCT resulting in dilation of diameter.[2]
In a study carried by Azab elsaied of Libya in 2017, pretreatment with olive leaf extract in 3 groups with a dose of 25, 50, and 100 mg per kg reduced significantly tubular necrosis and diameter of PCT. These histopathological alterations were caused by CCl4.[30] Said Elshama in 2016 showed that the use of 100 mg per kg per day naringin and 1.25 ml per kg per day olive oil significantly improved the cellular atrophy of PCT and DCT caused by 25 mg per kg of cyclosporin.[26] Ghorbel et al. from Tunisia in 2017 conducted one experimental study in which they used 300 ul of EVOO. It significantly reduced tubular dilation of PCT and DCT.[16]
Conclusion
The present study demonstrates that EVOO prevents the quantitative and qualitative histological changes caused by arsenic on kidneys which include the diameter of PCT and DCT, the diameter of glomeruli, width of Bowman’s space, and loss of brush border.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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