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VITAMINS C AND E AMELIORATE THE SODIUM ARSENATE INDUCED INTERSTITIAL FIBROSIS AND TUBULAR ATROPHY IN FETAL KIDNEYS OF ALBINO MICE.

Byline: QURESHI F., TAHIR M. AND LONE K. P.

ABSTRACT

Objectives: Epidemiological evidence indicates that exposure to arsenic contaminated drinking water during pregnancy may lead to various congenital abnormalities. The fetal renal tissue is impaired in relation to the developmental stage of kidney and to the amount and duration of arsenic exposure. This study evaluated the role of vitamin C and E in preventing the sodium arsenate induced mutilation of developing renal tissue of albino mice.

Materials and Methods: Albino female mice of BALB/c strain (24), were randomly divided into 4 groups after confirmation of pregnancy. The control group A1 received 0.1 ml/kg body weight distilled water; group A2 was given a single dose of sodium arsenate (35 mg/kg) body weight on 8th gestational day (GD) by intra-peritoneal (I/P) injection. The groups A3 and A4 were treated with sodium arsenate (35 mg/kg) body weight on 8th GD and vitamin C, (9mg/kg/day) and vitamin E (15 mg/kg/day) by I/P injections, from 8th GD for the rest of the pregnancy period. The fetal kidneys were extracted on 18th GD. Three coronal sections stained with PAS and Masson's trichrome from each kidney, were randomly chosen and examined for tubular injury and interstitial fibrosis.

Results: The mean scoring for histological changes in fetal kidney tubules/10 fields among various stu - dy groups was statistically significant (p < 0.000). Group A2 showed severe tubular necrosis and interstitial fibrosis confirmed by Masson's trichrome staining. Group A3 (vit C) and A4 (vit E) depicted mild to moderate improvement in tubular necrosis and interstial fibrosis.

Conclusions: This study supports the role of vitamin C and E in ameliorating the developing renal tissue from the oxidative stress induced by sodium arsenate.

Key Words: Antioxidants, interstitial fibrosis, sodium arsenate, [alpha]-tocopherol.

INTRODUCTION

Drinking water contamination with heavy metals has been associated with a risk of various congenital anomalies like limb defects, cardiac defects, chromosomal and urinary system anomalies.1 High levels of arsenic in drinking water have been correlated with the progressive chronic kidney disease.2 The mechanism of arsenic induced tissue injury is due to the production of reactive oxygen species(ROS) and free radicals which induce DNA damage in animals and humans.3 Antioxidants and antioxidant enzymes have a synergistic action in scavenging the free radicals and thus play a significant role in preventing many diseases like cancers, atherosclerosis, diabetes and neurodegenerative diseases.4

The interstitium of kidney consists of extravascular spaces of renal parenchyma, bounded on all sides by vascular and tubular basement membranes.5 The renal interstitium is involved in all functions of kidney and affects the GFR, growth and differentiation of parenchymal cells; therefore any changes in the interstitium points towards the renal disease.6 The destruction of renal tubules and interstitial capillaries with accumulation of extracellular matrix proteins results in interstitial fibrosis.7

Arsenic induced effects on animals and humans had been investigated by several researchers. Liu et al. (2000) demonstrated that cadmium administration accentuates the arsenic toxicity and caused a severe glomerulonephritis and interstitial fibrosis in mice kidneys.8 Chu et al. (2012) revealed that arsenic trioxide lead to cardiac fibrosisin guinea pigs.9 Ferzand et al (2008) investigated the histopathological changes in arsenic fed mice and establishedthe severe necrotic and degenerative changes in liver and kidney.10

The antioxidants like ascorbic acid and [alpha]-tocopherol prevents the lipid peroxidation and reduces the oxidative stress.11 The search for finding the cheap and effective measures for combating the arsenic toxicity was commenced by various researchers.12 Banerjee et al. (2009) reduced the arsenic induced fibrosis in liver with vitamin C.13 Pan et al. (2011) diminished the liver fibrosis in rats with grape seed extract which is rich in antioxidants.14 Umar (2007) used spinach extract to lessen the ROS and thus removed the arsenic burden in rats.15 This study aims to determine the antioxidant potential of vitamin C and E in preventing the interstitial fibrosis in developing kidneys of fetuses, exposed to arsenic through contaminated drinking water. These vitamins are freely available, inexpensive and can be used during pregnancy.

MATERIALS AND METHODS

Albino mice of BALB/c strain (twenty four females and eight males), 10 weeks old and weighing 30-35 gm were kept under controlled environments (temperature 22 +- 1AdegC) and humidity (40%-60%); in experimental research laboratory of University of Health Sciences Lahore. The animals were allowed to acclimatize for 7 days and were provided with standard rodent diet and distilled water ad libitum. The photoperiod was kept as 12 hour light/dark cycle. Three female mice were kept overnight with a single male for purpose of mating. The day, when vaginal plug appeared, was regarded as gestational day (GD) one. The pregnant females were randomly divided into four groups of six females each and placed in marked cages (n = 6). A1 served as a control and the other three (A2, A3 and A4) as experimental groups. Group A1 received 0.1 ml/kg body weight distilled water by I/P injection for 18 days.

Group A2 animals were injected with a single I/P injection of sodium arsenate (35 mg/kg body weight) dissolved in distilled water, on 8th day of gestation. Groups A3 and A4 females received sodium arsenate (35 mg/kg) on the 8th GD and vitamin C (9 mg/kg/ day) and E (15 mg/kg/day) by I/P injections respectively, from 8th day for the rest of the pregnancy period.16 The gestation period of mice is about 19 to 21 days and sodium arsenate was given on 8th day of gestation which is the start of embryonic period in which organogenesis takes place. Intra peritoneal injections given to avoid confounding factors of arsenate absorption by oral route and maximal transfer by placental circulation to fetuses. The doses were given according to previous literature.

The fetuses were extracted from the uterus by anaesthetizing the animals on 18th day of gestation to elude the risk of losing the fetal tissue by spontaneous abortions. The fetuses were dissected and the fetal kidneys were washed with distilled water and fixed in 10% formalin solution for histological study by standard procedures. Periodic acid - Schiff stained sections of 5 um thickness, were studied under a light microscope (Leica DM 1000) for thickening of basement membranes, mesangial cell proliferation and degenerative changes of tubules. The interstitial fibrosis was confirmed by Masson's trichrome staining. Three coronal sections from each kidney were randomly chosen and examined both qualitatively and quantitatively for the tubules and the interstitium. The observation bias was eliminated by coding the origin of group and it was un-coded after the results were interpreted.

Tubular Indices

The tubules were randomly selected from ten fields in corticomedullary region. They were assessed for cellular distension, cellular vacuolation and condition of their lumen as histological variables and scored as:

1 - 3 = Mild change

4 - 6 = Moderate change

7 - 9 = Severe change

Mean scores of histological changes were calculated. The frequency of histological changes in renal tubules was expressed in percentage.

Interstitial Indices

Interstitial fibrosis was estimated similarly as tubular injury score, in cortex and medulla separately and expressed as mild, moderate and severe. The interstitial fibrosis was confirmed by Masson's trichrome staining.

Statistical Analysis

The statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) version 18.0. Mean and standard deviations were calculated for the numerical variables. ANOVA was used to assess the mean difference among the groups. The post-hoc Tukey test was applied to assess the difference of means between the groups. The p value of a$? 0.05 was considered statistically significant.

RESULTS

The control group A1 showed the normal organization of renal corpuscles and tubules (Fig. 1a). The group A2 treated with sodium arsenate, illustrated the diffuse enlargement of glomerular tuft, capillary lumens were difficult to observe. There was proliferation of mesangial and endothelial cells and confluence of podocytes with parietal and tubular epithelium leading to obliteration of Bowman's spaces. Thickening of glomerular capillaries basement membranes were observed (Fig. 1b). Degenerative changes were observed in all segments of proximal convoluted tubules of group A2. The tubules also showed vacuolization and apoptotic changes and deposition of PAS +ve material on luminal surfaces (Fig. 1b).

In group A3, the renal corpuscles showed clear Bowman's spaces, glomerular capillaries had thin basement membranes (Fig 1c yellow arrow). Mesangial cell proliferation was not observed, the tubules showed the deposition of PAS +ve material on the luminal surface (Fig 1c). The group A4, demonstrated the extenuation of glomerular tuft but the mesangial cellularity was not observed, Bowman's spaces were clear, glomerular capillaries had thin basement membranes and endothelial thickening were not observed (Fig. 1d). The tubules showed the presence of PAS +ve material on the luminal surface (Fig. 1d).

Micrometric analysis of the tubular changes revealed moderate to severe tubular atrophy in group A2. Whereas, the groups A3 and A4 showed the reduction in tubular degenerative changes from mild to moderate. The data are shown in Table 1. The mean scoring for histological changes in fetal kidney tubules/10 fields among various study groups was statistically significant Table 2. The difference between the groups by test Tukey was significant among all groups (Table 2a).

The presence of interstitial fibrosis was confirmed by Masson's trichrome stain. Blue staining reaction was observed in group A2 where interstitial fibrosis was exhibited as patchy, striped pattern with tubular atrophy. There was preferential involvement of medulla (Fig. 2b). The groups A1 and A3 showed normal staining reaction, while group A4 depicted mild to moderate interstitial fibrosis (Fig. 2c and 2d), Table 1.

Table 1: Comparison of Tubular Histological changes/10 Fields from Mice Fetal Kidneys among Various Groups.

###A2 Sodium###A3 Sodium###A4 Sodium

###A1 Control

Tubular Injury Score###Arsenate###Arsenate + Vit. C###Arsenate + Vit. E###Total

###Freq###%###Freq###%###Freq###%###Freq###%

Mild change###40###100.0###-###-###40###100.0###7###17.5###54.4%

Moderate change###-###-###21###52.5###-###-###33###82.5###33.8%

Severe change###-###-###19###47.5###-###-###-###-###11.9%

Total###40###100.0###40###100.0###40###100.0###40###100###100.0%

Table 2: Comparison of Mean Scoring for Histological changes in Fetal Kidney Tubules per 10 Fields among Various Groups.

###A3 Sodium###A4 Sodium

###A1 Control###A2 Sodium

###Arsenate + Vit C###Arsenate + Vit E###p-

Parameter###(n = 40)###Arsenate (n = 40)

###(n = 40)###(n = 40)###value

###Mean###S.D###Mean###SD###Mean###SD###Mean###SD

Scoring of histological

###0.9###0.2###6.9###0.5###2.0###0.3###4.7###0.6###0.000*

changes in tubules.

Table 2a: Multiple Comparison of Mean Scoring for Histological changes in Fetal Kidney Tubules per 10 Fields among Various Groups according to Tukey Test.

###Comparison among Groups###Mean Difference###Level of Significance

###Groups ([alpha])###Group Compared ([beta])###([alpha]-[beta])###p-value

###(A2)###-6.0###0.000*

###(A1)###(A3)###-1.1###0.000*

###(A4)###-3.8###0.000*

###(A3)###5.0###0.000*

###(A2)

###(A4)###2.3###0.000*

###(A3)###(A4)###-2.7###0.000*

DISCUSSION

Sodium arsenate induces oxidative stress and increases the production of reactive oxygen species which induces the renal tissue damage. Nephrotoxicity of arsenic is due to its accumulation in the renal tissue during its excretion and thus affects the tubules.17 The current study encompassed the role of vitamins C and E as antioxidants in preventing the arsenic induced renal tissue impairment. In sodium arsenate treated group (A2) both glomerular and tubular damage was observed in histological preparations (Fig. 1b). The tubular damage was probably due to direct injury triggered by ROS and free radicals generated by sodium arsenate.18

Glomerulonephritis along with tubular atrophy with arsenic had been reported earlier by Liu et al. (2000).8

Vitamin C, due to its antioxidant potential, scavenges the free radicals from kidney and reduces the arsenic burden.19 Therefore, group A3 treated with sodium arsenate and vitamin C simultaneously showed marked improvement in mesangial cellularity, basement membranes became thin and tubular atrophic changes were also improved (Fig. 1c). Al-Attar (2011) described that supplementation of vitamin E along with heavy metals in mice minimizes the histological alterations in kidney and protect the organ from toxic agents and free radicals.20 Vitamin E neutralizes lipid peroxidation and unsaturated membrane lipids due to its oxygen scavenging effect.21,22 In this study, the group A4 injected with vitamin E along with sodium arsenate showed marked improvement in glomerular and tubular atrophic changes (Fig. 1d).

Micrometric analysis of tubular injury scores showed moderate to severe necrotic and degenerative changes in tubules of group A2 treated with arsenic only (Table 1). These effects of arsenic were attributed due to its physical accumulation in renal tissue and injury caused by ROS. Birri et al. (2010) gave male Wister rats different doses of arsenic in drinking water and demonstrated deleterious effects on tubules with periglomerular accumulation of arsenic along with copper.23 The tubular injury score among groups A3 and A4 showed marked improvements in tubular degenerative changes with simultaneous administration of vitamins C and E along with sodium arsenate respectively (Tables 1, 2, 2a). These effects of vitamin CandE were ascribed because these vitamins boost the antioxidant system by decreasing the arsenic burden in tissues and preventing the lipid peroxidation.11

Chinoy and Shah (2004) in their histocytometry study of kidney tubules and glomeruli revealed that administration of antidotes along with As2 O3 brought about a significant recovery in degenerative changes.24

The group A2 (arsenate only group) showed moderate to severe interstitial fibrosis (Fig. 2b) which was a result of direct toxic effects of sodium arsenate and due to the production of ROS which lead to increase in collagen production and thus affects the renal functions. Chu et al, (2012) demonstrated similar effects with As2 O3 causing cardiac fibrosis in guinea pigs.9 Liu et al (2000) had earlier established that combination of arsenic and cadmium produced massive renal interstitial fibrosis in mice.8 Children exposed to arsenic soiled drinking water in Chile were reported to have chronic lung disease and pulmonary interstitial fibrosis.25

The co-administration of vitamin C or E with arsenate in groups A3 and A4 respectively showed marked improvements in interstitial fibrosis (Fig. 2c and d). Sing et al. (2010) demonstrated in their study that arsenic exposed mice lungs showed necrosis and degenerative changes in bronchial epithelium and thickening of alveolar septa. These effects were antagonized by jaggery feeding which is rich in antioxidants.26 Yu et al. (2013) experimented with Chinese Dragon-Li cats and administered As2 O3 along with resveratrol, the protective effects of resveratrol increased the glutathione peroxidase activity and methylation of arsenic which is excreted in urine thus reducing the arsenic burden in renal tissue.27 The involvement of interstitial tissue and destruction of tubules with sodium arsenate was suggestive of the progressive kidney damage; however, vitamins C and E decreased the interstitial fibrous tissue, indicating recovery from arsenic induced injury.

This study concludes that vitamins C and E had ameliorated the sodium arsenate induced injurious effects on fetal renal tissue in albino mice.However, further studies on animals and humans are needed to understand the effects of arsenic exposure and arsenic contaminated drinking water on pre-conceptional and postconceptional periods.

Disclosure of Interest

The authors report no conflict of interest.

ACKNOWLEDGEMENTS

The authors acknowledge the laboratory support provided by the staff of department of anatomy of University of Health Sciences, Lahore.

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