Nephroprotective potential of selenium and taurine against mercuric chloride induced nephropathy in rats

Abstract

The study was aimed to estimate whether pre-treatment with sodium selenite or taurine would reverse kidney damage induced by intraperitoneal injection of mercuric chloride in rats. Animals were divided into six groups: (1) control group; (2) sodium selenite group; (3) taurine group; (4) HgCl₂ group; (5) sodium selenite pretreated group; (6) taurine pretreated group. The results demonstrated that HgCl₂ causes significant enhancement in serum malondialdehyde (MDA), creatinine, N-acetyl-beta-d-glucosaminidase (NAG), cystatin C, nephrin and interleukin 6 (IL-6) levels accompanied with significant reduction in serum nitric oxide (NO) level. Pretreatment with sodium selenite or taurine produces significant depletion in MDA, NAG, cystatin C, nephrin and IL-6 levels in concomitant with significant elevation in serum NO level as compared to HgCl₂ group. HgCl₂ induced pathological alterations in the kidney. The ultrastructural investigation of renal cortex of HgCl₂-administered group revealed that the glomerular basement membrane is uniform, the fenestrations of endothelial cells are swollen, and the secondary foot processes appear also swollen even fused at some points. The proximal convoluted tubules showed apical short and few microvilli, while, some tubular cells showed relatively normal microvilli. In contrast, sodium selenite or taurine pretreatment could significantly reduce the pathological alterations in the kidney caused by HgCl₂ intoxication. The current results suggested that selenium and taurine possess nephroprotective efficacy due to their antioxidative capacity and anti-inflammatory activity.

• Kidney function biomarkers
• kidney ultrastructure
• mercuric chloride
• rats
• sodium selenite
• taurine

Introduction

Mercury is one of the most widespread environmental pollutants which are used in industrial, pharmacological and agricultural fields.1 Mercuric chloride was formerly used in insecticides, batteries, as an antiseptic, disinfectant, preservative, in metallurgy, and as a photographic fixative.2 Mercury, both inorganic mercury (mercuric chloride) and methyl mercury (MeHg) may cause serious damage in various organs of the body systems via accidental and/or occupational exposures.3,4 Inorganic mercury is a well-established toxicant to human health which is found in water, food and air.5 Mercuric chloride is one of the most toxic forms of mercury because it easily forms organo-mercury complexes with proteins.6 It is well known as hepatotoxic,7 neurotoxic,8 nephrotoxic,9,10 hematotoxic11 and genotoxic.12 In addition, it is reported that mercuric chloride possesses adverse effects on the reproductive system in experimental animals.13

The kidney is the most important organ responsible for the excretion of unchanged drugs and/or their hydrophilic metabolites.14 The primary target organ for inorganic salts of mercury is the kidney. The symptoms of nephrotoxicity caused by mercury in man and experimental animals reflect the renal function defects largely concentrated in the proximal tubules. The pathophysiological symptoms caused by mercury include phosphaturia, glucosuria, ketonuria, proteinuria, and wasting of various cations.15 Heavy metals form metal-binding complexes when bind with the protein of the plasma membrane,16 and once the complex is formed the heavy metal causes cellular damage in the respective tissue. Mercury poisoning leads to renal insufficiency in various animals through the way of forming these types of complexes.17

The toxicity of mercuric compounds is thought to be due to their high affinity for cysteine thiol and generation of reactive oxygen species (ROS), which usually leads to oxidative stress and cellular damage18 in addition to the alteration in the antioxidant enzyme activities in different organs and tissues.19

Selenium is an essential element and well-established antioxidant for all tissues of animals and human. It is a structural component of several antioxidant enzymes including glutathione peroxidases and thioredoxin reductase.20 Sodium selenite, a common dietary form of selenium, is recognized as essential in animal and human nutrition.21 Selenium is known to have the ability to reduce the toxicity of several xenobiotics including heavy metals.19 Selenium is known to possess an affinity constant for mercury higher than that of sulfhydryl compounds and the protection offered by sodium selenite in mercury intoxication has been attributed to the formation of Hg–Se–S complex which is stated to be non-toxic.7

Taurine is a sulfur-containing amino acid that conjugates with bile acids in the liver and chemically similar to acetylcysteine, an agent for treating the heavy metal intoxication.22 Taurine plays an important role in reducing the toxic effect of copper, lead, cadmium, oxidized fish oil, oxidized cholesterol and vitamin A in rats.23–26 In addition to its important physiological role in osmoregulation, bile acid conjugation, modulation of the central nervous system, cell proliferation, viability and prevention of oxidant induced injury in many tissues.27,28 The beneficial effect of taurine as an antioxidant in biological system has been attributed to its ability to stabilize biomembranes and also scavenging ROS in animals.29,30 The present work was designed to assess the efficacy of pretreatment with selenium and taurine in the protection of kidney against mercuric chloride-induced nephropathy in rats.

Materials and methods

Chemicals

HgCl₂, sodium selenite, taurine (2-aminoethane sulfonic acid) and all other chemicals were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO) standard commercial supplier.

Experimental animals

Forty-eight adult male Wistar albino rats weighing 120–130 g were obtained from Animal House Colony of Helwan Farm, Cairo, Egypt. The animals were housed under standard laboratory conditions (12 h light and 12 h dark) in a room with controlled temperature (24.3 °C) during the experimental period. The rats were provided ad libitum with tap water and fed with standard commercial rat chow. Animal procedures were performed in accordance with Guidelines of Ethical committee for the Care and Use of Animals at the National Research Centre, Egypt.

Experimental design

After one week of acclimatization, animals were divided into six groups (8 rats/group). Group 1: served as control, received distilled water intraperitoneally once daily for 15 days and left untreated for other 15 days. Group 2: sodium selenite group received 0.1 mg/kg b.wt sodium selenite by gastric gavages once daily for 30 days.31 Group 3: taurine group received 50 mg/kg b.wt taurine by gastric gavages once daily for 30 days.32 Group 4: mercuric chloride (HgCl₂) group received 0.5 mg/kg b.wt HgCl₂ intraperitoneally once daily for 15 days31 and left untreated for other 15 days. Group 5: sodium selenite-treated group received 0.1 mg/kg b.wt sodium selenite orally by gavages once daily for 15 days prior HgCl₂ and for other 15 days in concomitant with HgCl₂. Group 6: taurine treated group received 50 mg/kg b.wt taurine orally by gavages once daily for 15 days prior HgCl₂ and for other 15 days in concomitant with HgCl₂.

At the end of the experimental period (30 days), rats were fasted overnight, and then anesthetized with diethyl ether and the blood samples were collected by direct puncture of the right ventricle and from the retro-orbital venous plexus, in sterile separator tubes. The blood samples were centrifuged at 1300 × g for 15 min to separate sera for assessment of biochemical parameters. After blood collection, kidney samples were dissected immediately, washed with sodium phosphate buffer and prepared for light and electron microscopic investigations.

Biochemical analyses

Serum lipid peroxidation represented by malondialdehyde (MDA) was estimated by spectrophotometric method33 using commercial kits (Biodiagnostic reagent kits, Egypt). Serum nitric oxide (NO) represented by nitrite was determined according to the method of Granger et al.34 using a Roche nitrite/nitrate assay kit. The nitrate present in the sample is reduced to nitrite by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of the enzyme nitrate reductase. The nitrite formed reacts with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride to give a complex which was measured at 550 nm. The coefficient of variation is 15%, with lowest detectable border of 3.0 mmol/mL. Serum creatinine was analyzed by colorimetric method of Heinegard and Tiderstrom35 using commercial kits (LABTEST, Diagnostica S.A, Minas Gerais, Brazil). Serum interleukin-6 (IL-6) and cystatin C were determined using ELISA kit according to the manufacturer’s protocol (Glory Science Co., Ltd., Veterans Bivd., CA). Serum N-acetyl-beta-d-glucosaminidase (NAG) activity was estimated using the commercially available NAG assay kit from Diazyme Laboratories (Poway, CA) according to the method of Yuen et al.36 Serum nephrin was measured using a commercially available ELISA kit according to the manufacturer’s protocol.

Histological examination

For histological investigation of kidney, each left kidney of rats in the different studied groups was divided into two halves, one for the light microscopic examination, while a small cortical piece of the other half was processed for the electron microscopic study. The spacemen's for light microscopic examination were preserved in 10% neutral buffered formalin for 24 h,37 then dehydrated in alcohols (70%, 90% and absolute), cleared in benzene and embedded in paraffin wax and 5 µm thick sections were stained with hematoxylin and eosin (H&E) stains.38 The samples for electron microscopic study were fixed in 3% glutaraldehyde in phosphate buffer for 24 h, washed in phosphate buffer for 20 min (3 changes), then a second fixation was performed in 1% buffered osmium tetroxide for 1.5 h. Specimens were washed again in phosphate buffer, dehydrated in ascending grades of alcohol and embedded in pure fresh resin. Ultrathin sections (80–90 nm) were cut on Leica ultramicrotome, stained with uranyl acetate and lead citrate39 and examined by Joel Jem transmission electron microscope at 60 kV accelerating voltage In the Regional Center for Mycology and Biotechnology Al-Azhar University.

Statistical analysis

The data were analyzed using version 11.0 of the computer-based statistical product and service solutions (SPSS, 2001, Chicago, IL). All the data are expressed as mean ± standard error of mean. Analysis of the data was done using one-way analysis of variance (ANOVA) to detect the significant difference among the studied groups. A level of p < 0.05 was defined as statistically significant. Pearson's correlation coefficient was used to determine significant correlation between serum NAG, cystatin C and nephrin concentrations and creatinine levels.

Results

Effect of pretreatment with sodium selenite and taurine prior HgCl₂ on serum MDA and NO levels

The data in Table 1 illustrated that animals administered with HgCl₂ show significant increase (p < 0.01) in serum MDA level as compared to the control group. Pretreatment with sodium selenite or taurine prior HgCl₂ reduced the process of lipid peroxidation as indicated by the significant decline (p < 0.01) in serum MDA level as compared to HgCl₂ group. The results in Table 1 also indicated that, administration with HgCl₂ results in significant decrease (p < 0.01) in serum NO level as compared to the control group. In contrast, animals pretreated with sodium selenite or taurine prior HgCl₂ showed significant increase (p < 0.01) in serum NO level as compared to HgCl₂ group. Noteworthy, treatment with sodium selenite or taurine alone produced insignificant change (p > 0.05) in serum MDA and NO levels when compared with the control group.

Table 1. Effect of pretreatment with sodium selenite and taurine prior HgCl₂ on serum MDA and NO levels.

Groups	Serum MDA (nmol/mL)	Serum NO (mmol/mL)
Control group (−ve control group)	4.948 ± 0.208^a	1.395 ± 0.185^a
Sodium selenite group (+ve control group)	4.944 ± 0.179^a	0.992 ± 0.208^a
Taurine group (+ve control group)	5.178 ± 0.175^a	1.063 ± 0.192^a
HgCl2 group	6.774 ± 0.359**^b	0.524 ± 0.047**^b
Sodium selenite + HgCl2 group (treated group)	4.487 ± 0.361**^c	1.298 ± 0.190**^c
Taurine + HgCl2 group (treated group)	4.303 ± 0.410**^c	1.232 ± 0.152**^c

Note: Data are presented as mean ± SE of the eight animals/group. **p < 0.01.

^aSignificant difference between positive groups and negative control group.

^bSignificant difference between HgCl₂ group and negative control group.

^cSignificant difference between treated groups and HgCl₂ group.

Within each column, means superscript with the same letter are not significantly different.

Effect of pretreatment with sodium selenite and taurine prior HgCl₂ on serum creatinine level, NAG activity, cystatin C and nephrin levels

Results in Table 2 revealed that, serum creatinine level, NAG activity, cystatin C and nephrin concentrations were significantly increased (p < 0.001 and p < 0.01, respectively) in rats administered with HgCl₂ when compared with the control group. However, treatment with sodium selenite or taurine alone did not show any significant alteration (p > 0.05) in serum creatinine level, NAG activity, cystatin-C and nephrin concentrations as compared to the control group. Whereas, a significant decrease (p < 0.001, p < 0.01 and p < 0.05) in serum creatinine level, NAG activity, cystatin C and nephrin activity concentrations were recorded in the groups pretreated with sodium selenite or taurine prior HgCl₂ compared with HgCl₂ group. By comparing between the groups of animals pretreated with sodium selenite and those pretreated with taurine, it has been found that serum NAG activity was significantly decreased (p < 0.01) in animals pretreated with sodium selenite compared to those pretreated with taurine (Table 2). The panels in Figure 1 showed that serum creatinine level displayed a positive correlation with serum NAG (r = 0.73; p = 0.000) activity, cystatin C (r = 0.694; p = 0.000) and nephrin (r = 0.506; p = 0.002) levels.

Figure 1. Pearson’s correlation coefficient between serum-creatinine level and serum N-acetyl-beta-d-glucosaminidase activity, cystatin C and nephrin level.

Table 2. Effect of pretreatment with sodium selenite and taurine prior HgCl₂ on serum creatinine levels, N-acetyl-beta-d-glucosaminidase activity, cystatin C and nephrin levels.

Groups	Serum creatinine (mg/dL)	Serum NAG (IU/L)	Serum cystatin C (μg/L)	Serum nephrin (pg/mL)
Control group (−ve control group)	2.575 ± 0.175^a	16.92 ± 0.339^a	44.596 ± 1.31^a	0.471 ± 0.0263^a
Sodium selenite group (+ve control group)	2.56 ± 0.327^a	19.979 ± 1.643^a	43.148 ± 0.951^a	0.515 ± 0.0145^a
Taurine group (+ve control group)	2.45 ± 0.130^a	20.02 ± 2.102^a	44.051 ± 1.112^a	0.471 ± 0.0132^a
HgCl2 group	8.25 ± 0.137***^b	33.51 ± 2.086***^b	53.615 ± 4.2**^b	0.564 ± 0.0091**^b
Sodium selenite + HgCl2 group (treated group)	3.475 ± 0.226***^c	20.24 ± 2.218**^c,^d	45.124 ± 3.673**^c	0.511 ± 0.0217*^c
Taurine + HgCl2 group (treated group)	3.675 ± 0.211***^c	25.561 ± 0.560**^c	44.209 ± 2.46**^c	0.504 ± 0.0153**^c

Note: Data are presented as mean ± SE of the eight animals/group. *p < 0.05, **p < 0.01 and ***p < 0.001.

^aSignificant difference between positive groups and negative control group.

^bSignificant difference between HgCl₂ group and negative control group.

^cSignificant difference between treated groups and HgCl₂ group.

^dSignificant difference between sodium selenite treated group and taurine treated group.

Within each column, means superscript with the same letter are not significantly different.

Effect of pretreatment with sodium selenite and taurine prior HgCl₂ on serum IL-6 level

Serum IL-6 level in HgCl₂ administered rats showed significant increase (p < 0.001) as compared to the control group. No significant alteration (p > 0.05) in serum IL-6 production was detected in rats treated with sodium selenite or taurine alone when compared with the control group. Conversely, there was significant decline (p < 0.001) in serum IL-6 level in sodium selenite or taurine pretreated groups as compared to HgCl₂ group. Serum IL-6 level decreased by 56.04% in taurine pretreated group while, it decreased by 45.3% in sodium selenite pretreated group (Table 3).

Table 3. Effect of pretreatment with sodium selenite and taurine prior HgCl₂ on serum IL-6 level.

Groups	Serum IL-6 (ng/L)
Control group (−ve control group)	2.209 ± 0.192^a
Sodium selenite group (+ve control group)	1.971 ± 0.303^a
Taurine group (+ve control group)	2.188 ± 0.178^a
HgCl2 group	5.03 ± 0.358***^b
Sodium selenite + HgCl2 group (treated group)	2.752 ± 0.272***^c
Taurine + HgCl2 group (treated group)	2.211 ± 0.332***^c

Note: Data are presented as mean ± SE of the eight animals/group. ***p < 0.001.

^aSignificant difference between positive groups and negative control group.

^bSignificant difference between HgCl₂ group and negative control group.

^cSignificant difference between treated groups and HgCl₂ group.

Within each column, means superscript with the same letter are not significantly different.

Light microscopic results

Light microscopic examination of kidney cortical sections of control group showed the renal corpuscles formed of Bowman's capsule surrounded the glomerular capillary tuft. The Bowman's capsule was formed of parietal and visceral layers. The proximal convoluted tubules (PCTs) were also seen and lined by simple cuboidal epithelium with acidophilic cytoplasm. The epithelium nearly occluding the lumina, few numbers of cells per cross-section could be noticed. The distal convoluted tubules (DCTs) were lined by simple cuboidal epithelium. The number of cells per cross-section was more than those of the PCTs (Figure 2a). Renal cortical sections of sodium selenite or taurine group were more or less similar in histological structure to those of control group (Figure 2b, c). The renal cortex of HgCl₂ group showed that the renal corpuscle formed of Bowman’s capsule surrounded the glomerular capillary tuft. The PCTs were the most affected structure in the cortex. Most of the PCT lined by low cuboidal cells, some lumina appeared wider and contained acidophilic masses, some tubules were denuded or contained detached or vacuolated or disintegrated cells (Figure 2d). While, examination of renal cortex of sodium selenite pretreated group (sodium selenite + HgCl₂) showed that the renal corpuscles and most of the tubules were similar to the control group. Some areas appears vacuolated and contained mononuclear cellular infiltration, few tubules contained disintegrated cells between its lining epithelium even some detached cells could be noticed in the lumen, few tubules contained small pale acidophilic mass (Figure 2e). Also, the renal cortex of taurine pretreated group (taurine+HgCl₂) showed that the renal corpuscles were similar to those of the control group. Most of the cortical tubules were similar to those of the control group. Few tubules contained vacuolated or detached cells between their lining epithelium, few tubules contained small acidophilic masses, cellular infiltration was also noticed (Figure 2f).

Figure 2. Photomicrographs of kidney sections stained by H & E. (a): A section taken from kidney of control group, (b): a section taken from kidney of sodium selenite treated group shows 1-PCT, 2-DCT, 3-glomerular capillary tuft, 4-parietal layer of Bowman’s capsule, (c): a section taken from kidney of taurine treated group shows 1-glomerular capillary tuft, 2-parietal layer of Bowman’s capsule, 3-PCT, 4-DCT, (d): a section taken from kidney of HgCl₂ group shows 1-glomerular capillary tuft, 2-parietal layer of Bowman’s capsule, 3-intraluminal acidophilic mass, 4-denuded tubule with detached cell, (e): a section taken from kidney of sodium selenite pretreated group shows 1-glomerluar capillary tuft, 2-PCT, 3-PCT lined by normal and degenerated cell (5), 4-interaluminal small mass, 6-cellular infiltration and (f): a section taken from kidney of taurine pretreated group shows: 1-rernal tubule contained between its lining cells some vacuolated cells, 2-normal PCT, 3-cellualar infiltration, 4-small luminal acidophilic mass (H&E X200).

The electron microscopic results

In control group, the examination of ultrathin sections of renal cortex revealed the usual components of the glomeruli. Several capillary loops were recognized by their content of erythrocytes and precipitated plasma proteins. The capillaries were lined by a thin layer of fenestrated endothelium. Mesangial cells are usually present between the capillary loops to support them. Podocyte and its primary process that gives rise to numerous secondary foot processes could be seen. The glomerular basement membrane (BM) separated the podocytes from the capillary endothelium. The thickness of the BM was uniform. The fenestrated capillary endothelium was closely applied to luminal surface of the glomerular BM. On the opposite side the numerous secondary foot processes were seen rested on the BM (Figure 3a). The PCT was characterized by its narrow lumen. The large cubical cells have prominent apical microvilli. The cells were closely related to each other due to the interdigitation junction between them. The nuclei were rounded and small in relation to the large sized cells. Numerous scattered mitochondria and few lysosomes and some pinocytic vesicles were observed. At the tubular cells basal infoldings could also be recognized (Figure 3b,c). Examination of ultrathin sections of sodium selenite or taurine group showed no variation from the control group (Figure 3d,e). Ultrathin kidney sections of HgCl₂ group showed that the glomerular BM was uniform, the fenestrations of endothelial cells were swollen, and the secondary foot processes appeared also swollen even fused at some point (Figure 4a). The PCT showed apical short and few microvilli (Figure 4b), some tubular cells still have relatively normal microvilli. Some of the epithelial proximal cells were ruptured and their contents expelled out into the lumen, some of mitochondria were swollen and fused, some myeloid bodies could be seen, numerous vacuoles and lysosomes could also be noticed (Figure 4c,d). In sodium selenite pretreated group (sodium selenite + HgCl₂), ultrathin sections showed that the glomerular BM, fenestrations of endothelial cells and secondary foot processes showed no variation from the control group (Figure 5a). The PCT showed broad similarity to the control, microvilli and many mitochondria similar to control, lysosomes and vacuoles were more numerous than those of the control (Figure 5b). Kidney ultrathin sections of taurine pretreated group (taurine + HgCl₂) showed that the glomerular BM, fenestrations of endothelial cell and secondary foot processes were quite similar to the control group (Figure 5c). The PCT showed prominent improvement than the HgCl₂ group. In spite of that the microvilli and many mitochondria were similar to the control, some mitochondria still swollen, numerous lysosomes and vacuoles could be seen in Figure 5(d).

Figure 3. From kidney of control rat, (a): shows many capillary loops (C), MN: mesengial cell nucleus, PN: podocyte nucleus, RBC: red blood corpuscle. Upper right rectangle showing: 1-indothelial fenstrations, 2-BMP2: secondary podocytic foot processes (O.M. X10,000 & X25,000), (b): shows part of the PCT, N: nucleus of tubular cells, 1: pinocytic vesicle, 2: lysosome, 3: basal infolding, 4: lateral interdigitation, Mv: microvilli. Upper right square is a magnification of a microvilli (X 5000 & 8000), (c): shows part of PCT, N: nucleus of tubular cell, 1: mitochondria, 2: basal infoldings (X10,000), while (d): section obtained from kidney of sodium selenite treated group shows part of PCT, 1: mitochondria, 2: basal infoldings, N: nucleus of tubular cell (X10,000), and (e): section obtained from kidney of taurine treated group shows part of PCT, 1: mitochondria, 2: basal infoldings, 3: pinocytic vesicle, N: nucleus of tubular cell, Mv: microvilli (X8000).

Figure 4. Electron micrographic ultrathin sections obtained from kidney of HgCl₂ group. (a): Shows many capillary loops (c), RBC: red blood corpuscle. Upper left rectangle showing higher magnification of: 1-BM, 2-endothelial fensterations, P2: secondary foot processes (O.M. X12,000 & X25,000), (b): shows part of PCT. Arrow showing short few microvilli (X5000), (c): part of PCT: 1: edematous fused mitochondria, 2: ruptured cell which expels its contents in the lumen, 3: many vacuoles, 4: myeloid body, 5: lysosome, N: nuclei of tubular cells (X4000), (d): part of PCT: 1: myeloid body, 2: lysosome, 3: swollen fused mitochondria, Mv: relatively normal microvilli (X8000).

Figure 5. Electron micrographic ultrathin sections of renal cortex. (a,b): Sections obtained from kidney of sodium selenite pretreated group, (a): shows many capillary loops (c), PN: podocytic nucleus, RBC: red blood corpuscle. Lower right rectangle showing: 1-BM, 2-endothelium fenstrations, P2: secondary foot processes. (O.M. X10,000 & X25,000), (b): shows part of the PCT. 1-mitochondria, 2-lysosome, 3-vaculoes, N: nucleus of tubular epithelial cell, Mv: microvilli (X5000). (c,d): Sections obtained from kidney of taurine pretreated group, (c): shows many capillary loops, RBC: red blood corpuscle. Upper left rectangle showing: 1-fenstration of endothelium, 2-BM, P2: secondary foot processes (X10,000 & X25,000) and (d): shows part of PCT: 1-microvilli, 2-vacuole, 3-normal mitochondria, 4-lysosome, 5-swollen mitochondria (X6000).

Discussion

In the present study, animals administered with HgCl₂ showed significant increase in serum MDA level, while the pretreatment with sodium selenite or taurine prior HgCl₂ administration significantly reduced the serum level of MDA. These findings are in agreement with those of Rao and Chhunchha4 and Sheikh et al.40 who demonstrated significant increase in lipid peroxidation level in HgCl₂ treated groups as compared to the control. Also, Sharma et al.9 recorded a time-dependent significant enhancement in MDA level after HgCl₂ administration. Mercury promotes the formation of ROS such as hydrogen peroxides and highly reactive hydroxyl radical by Fenton transition equation, and enhances the subsequent iron and copper-induced production of lipid peroxides.7 Lipid peroxides alter membrane structure and are highly disruptive of mitochondrial structure and contribute to nephrotoxicity.

Guzzi et al.41 found that selenium reverses the elevated levels of thiobarbituric acid-reactive substances upon exposure to mercuric chloride in mice. Draz et al.42 found that there is significant increase in serum MDA level in the workers in lamp factory exposed to heavy metals (mercury, lead and cadmium) in comparison with the unexposed control group. Following treatment with selenium and vitamin E, they demonstrated significant decrease in serum MDA level in workers after two months of treatment in comparison with themselves before treatment. Hence, it was suggested a possibility of the protective role of selenium against mercury toxicity, via the formation of Hg–Se complexes, the redistribution of Hg, the competition for binding sites, metabolism of toxic forms of Hg and prevention of oxidative damage.

Deng et al.43 reported that, as compared to the MeHg-exposed group, the content of MDA of taurine treated group is decreased significantly. Samipillai and Jagadeesan32 stated that during the recovery period of mercuric chloride intoxicated rats, the treatment with taurine for 15 days results in remarkable recovery of the animals from the adverse effects of mercury toxicity. This finding has been documented by the restoration of lipid peroxidation level and the antioxidant enzymes activity to near normal valves in mercury intoxicated animals. Yu et al.44 stated that, in rats pretreated with taurine, the contents of MDA in kidney are significantly decreased compared with those treated with mercury. In general, elevation in the levels of lipid peroxide has been observed in mercury intoxicated animals compared to that of normal and taurine treatment could ameliorate the elevation and retains the values close to normal.45 These results together with our results suggested that taurine plays a vital role to reduce the toxic effect of mercury in the kidney tissue of rats. The proposed mechanism for the role of taurine in kidney protection is that taurine functions as a direct antioxidant that scavenges oxygen free radicals, thus inhibiting lipid peroxidation, and as an indirect antioxidant that prevents the increase in membrane permeability resulting from oxidative injury in kidney.46 As an indirect antioxidant, taurine has been considered as a membrane stabilizer that can maintain the membrane organization, prevent ion leakage and water influx and subsequently avoid cell swelling.47 Taurine is a special amino acid, which possesses an amino group and a sulfonate group. These functional groups might bind with heavy metal and stimulate the excretion of such compounds. By this way, taurine may play a vital role to reduce the toxic effect of mercury in the kidney of rats.25

The present results also indicated that, administration with HgCl₂ resulted in a significant decrease in serum NO level, while animals pretreated with sodium selenite or taurine showed significant increase in serum NO level. These results are in accordance with El-Boshy and Taha48 who stated that mercuric chloride treated group shows significant decrease in serum NO level when compared with the control. Berkels et al.49 and Santarelli et al.50 revealed that plasma NO concentration is significantly lower in mice treated with the higher dose of mercury than in the control group. Huang et al.51 observed that, NO levels in the brain are significantly decreased in all HgCl₂-treated groups. Methyl mercury was found to inhibit the production of induced NO and nitric oxide synthase from human alveolar macrophages.52 Lu et al.53 demonstrated an enhancement of relaxation in aortic rings from rats after receiving selenium supplement. They explained this finding as selenium can increase the release of NO. Hagar et al.54 revealed that taurine treatment prevents the decrease in NO levels induced by cyclosporine A administration. Moreover, supplementation with l-arginine, the NO precursor, prevents cyclosporine A-induced endothelial dysfunction.55

Agarwal et al.56 reported that exposure to mercury significantly increases serum creatinine level. Sarwar Alam et al.57 and El-Shenawy and Hassan58 stated that HgCl₂ treatment alone raises the levels of serum creatinine by about 3.19 folds as compared to saline treated control. These findings supported the present results of elevating serum creatinine level in HgCl₂ administered rats. The current results revealed that the pretreatment with sodium selenite prior HgCl₂ produces significant decrease in serum creatinine level. Guzzi et al.41 found that selenium reverses the elevated levels of serum creatinine upon exposure to mercury chloride in mice. Erdem et al.59 and Ahmad et al.60 revealed that taurine treatment reduces gentamicin and potassium bromate-induced increase in serum creatinine level. This result supports our finding as the pretreatment of taurine prior HgCl₂ resulted in significant reduction in serum creatinine level.

NAG is a lysosomal enzyme present in high concentrations in renal proximal tubular cells. In the present study, serum NAG concentration was significantly increased in rats injected intraperitoneally with HgCl₂. Whereas, a significant reduction in NAG activity was recorded upon pretreatment of selenium or taurine prior HgCl₂ administration. These results are in accordance with those of Abdalla et al.61 who reported that there is an increase in NAG activity in the kidney and urine of MeHg-treated group, when compared with the control group. The excretion of urinary NAG indicates the occurrence of tubular cell damage in the MeHg-treated group as the increase in urinary NAG excretion is one of the most sensitive markers for renal disease and suggests proximal tubule injury.62 Jarosinska et al.63 revealed a statistically significant increase in the NAG activity in workers highly exposed to mercury vapor, indicating mercury toxicity in proximal tubular cells. Girolami et al.64 reported that, mercuric chloride exposure is associated with an increased release of the lysosomal enzyme NAG.

El-Demerdash65 observed that, when selenium in the form of sodium selenite is given in combination with mercury administration, it significantly modulates the toxic effects of mercury on various enzymatic activities in rats. Yu et al.66 revealed that urine activities of NAG decreased significantly in rats pretreated with taurine prior to mercury administration as compared with those injected with mercury alone. Thus, pretreatment with taurine displayed certain protection against acute nephrotoxicity caused by mercury.

Cystatin C has been known as a marker of early stage of chronic renal failure. Cystatin C has been advocated as a better marker of glomerular filtration rate (GFR) than serum creatinine because, unlike creatinine, it is not secreted by the renal tubule, and it is independent upon muscle mass, age or gender, and does not have a circadian rhythm.67 In the present study, serum cystatin C concentration was significantly increased in rats injected intraperitoneally with HgCl₂ with respect to control animals. Whereas, a significant decrease in serum cystatin-C level was noticed in the groups of rats pretreated with sodium selenite or taurine prior HgCl₂. These results are in accordance with those of Bin et al.68 who found a significant increase in serum cystatin C in rabbits intoxicated with HgCl₂ as compared to the control group. Ozer et al.69 demonstrated that serum cystatin C level is a sensitive and specific marker of various types of nephrotoxicant-induced lesions in the kidney.

Brandao et al.70 stated that selenium supplementation is the best way to restore renal toxic effects induced by mercuric chloride in mice. Selenium is known to protect against nephrotoxicity induced by mercury in experimental studies and in the populations with low selenium levels who may be more vulnerable to mercury toxicity.71 Deng et al.72 reported that taurine can effectively counteract the adverse effects of methylmercury. Jagadeesan and Sankar-Samipillai30 suggested that taurine plays a vital role in reducing the mercury toxicity in intoxicated animals.

Nephrin is a podocyte cell adhesion protein located at the slit diaphragm area of the kidney glomerulus. In the present study, serum nephrin concentration was significantly increased in rats injected intraperitoneally with HgCl₂ with respect to control animals. While, a significant decrease in serum nephrin level was recorded in rats pretreated with sodium selenite or taurine prior HgCl₂. Son et al.73 observed that both serum and urine concentrations of nephrin were significantly higher in the sever preeclamptic group than in the normal pregnancy group. They explained this result as that preeclampsia is associated with renal pathologic changes as glomerular endotheliosis. Pfeilschifter74 reported that in inflammatory kidney diseases like the mesangioproliferative glomerulonephritis, nephrin expression is upregulated.

In the present study, serum IL-6 level of HgCl₂ administered rats showed significant increase as compared to the control group. While, there was significant decline of serum IL-6 level in sodium selenite or taurine pretreated groups as compared to HgCl₂ administered group. These results are in agreement with those of Yasutake and Nakamura75 who stated that plasma IL-6 level is markedly elevated in inorganic mercury or methyl mercury-administered mice. Kempuraj et al.76 found that HgCl₂ stimulates IL-6 release from human umbilical cord blood derived cultured mast cells compared to untreated cells. It has been found that high doses of Hg²⁺ induce inflammatory status with consequent Th1/Th2 imbalance50 and eventual development of neoplasm77 or autoimmune disease.78 The mechanisms by which Hg²⁺ affects the cell-mediated immune response are still unclear. Moreover, Chang and Tsai79 reported that MeHg causes IL-6 production and cytotoxicity simultaneously and Nechemia-Arbely et al.80 found that administration of HgCl₂ to mice results in rapid and substantial elevation in serum IL-6 level concurrent with the development of acute kidney injury. Tseng et al.81 demonstrated that serum selenium is inversely associated with inflammatory cytokine (IL-6) among elderly living in long-term care facilities in Taiwan. Shilo et al.82 observed that selenium supplementation strongly attenuates the blood IL-6 elevation. This differential effect of selenium may be one of the mechanisms underlying its anti-inflammatory activity. The exact mechanisms by which selenium enhances immune function are not fully known. It is likely that selenium exerts its effect by altering the redox status of the cells83 or by meeting the increased requirements for selenoproteins of the activated immune cells. Upregulation of selenophosphate synthetase activity, directed towards the synthesis of selenocysteine, a key constituent of selenoproteins, in activated T cells84 and upregulation of several protein biosynthesis genes in the lymphocytes of subjects given selenium supplementation have been previously reported.85

Zhuo et al.86 showed that combination therapy with taurine, epigallocatechin gallate and genistein decreases the levels of serum IL-6 in rats with hepatic fibrosis induced by alcohol. Schuller-Levis and Park87 reported that supplementation with taurine in the drinking water increases the available taurine both systemically and at the site of inflammation and downregulates proinflammatory cytokine production. Thus, taurine may provide a useful prophylactic approach to preventing tissue damage resulting from inflammation.

The light microscopic examination of renal cortex of mercuric chloride group showed PCTs were the most affected part; some tubules were denuded or lined by disintegrated cells. The PCTs represent the main bulk of the renal cortex since the ratio of cross-sections of proximal to distal tubules surrounding any renal corpuscle is usually 7:1.88 These results are in agreement with those of Deepmala et al.89 who mentioned that the exposure to mercury leads to degeneration of renal tubules with obstructed lumen. Renal tubular cell injury is the primary pathogenetic event in both ischemic and nephrotoxic acute renal failure. Weinberg et al.90 described the morphological changes of the kidney of rats exposed to HgCl₂ by focal loss of brush border membrane of tubular cells, associated by an increase in small vacuoles and small dense bodies as shown in the electron microscopic investigation. Fowler91 mentioned that HgCl₂ produces number of effects on the renal proximal tubular cells indicated by marked blebbing and exfoliation of brush border membrane as an early sign of exposure, and they explained the cause of cell death after exposure to mercury by massive calcium influx into cells causing its death.

The electron microscopic examination of the kidney of HgCl₂ group showed that the endothelial cells and secondary podocytic processes are edematous and swollen, while the PCT showed short, few and small microvilli, swollen, and fused mitochondria. Many vacuoles and myeloid bodies were also seen. It is clear from histopathological observation that mercury causes necrosis of tubular epithelium along with several other degenerative lesions. These observations have been sustained by reports of other investigators with various metals as aluminum and beryllium administration.92,93 Joshi et al.94 supported these findings and they stated that the heavy metals induce toxicity through oxidative stress, and the toxicological profile of mercury is associated with a variety of biochemical abnormalities as loss of functional integrity of cell membrane. Takeda et al.95 tried to explain the cause of podocytic processes abnormalities by the podocalyxin/Na⁺, H⁺ exchange regulatory factor2/ezrin/actin interactions which are disrupted in pathologic conditions associated with changes in glomerular epithelial cell foot processes, indicating their importance for maintaining the unique organization of this epithelium. In the present study, the microscopic investigation of kidney of HgCl₂ administered rats revealed that the microvilli appear small, few and short. Stevens and Lowe96 stated that the microvilli are finger like projections of the apical cell surface which are maintained by a bundle of actin filament that forms a core running through each microvillus. In case of renal injury whatever toxic or ischemic injury there is cytoskeleton disruption which leads to cellular disconfiguration including microvilli.97

Yamada98 studied the mechanism of renal damage induced by several nephrotoxic compounds, and stated that the formation of myeloid bodied is one of the signs of toxicity. Sundin et al.99 explained the mechanism by which myeloid bodies are formed. The inhibition of lysosomal phospholipases leads to subsequent accumulation of phospholipids and formation of myeloid bodies which are implicated as a direct mechanism of nephrotoxicity. Underwood100 stated that the mitochondria play a key role in intracellular oxygen metabolism. Any chemical agent that depletes ATP either by interfering with mitochondria oxidative phosphorylation or by consuming ATP in its metabolism will compromise the integrity of the membrane pumps and expose the cell and its organelles to the risk of lysis. Deepmala et al.89 concluded that the dimethyl mercury induces tissue damage due to oxidative stress and generation of ROS. Nony and Schnellmann97 reported that the injury of the PCTs whatever toxic or ischemia is characterized by mitochondrial dysfunction and ATP depletion.

Cellular infiltration usually excites or occurs as a result of tissue injury in order to eliminate the destructed tissues in the way for tissue restoration.101 D'Amico et al.102 reported that the cellular infiltration is most probably directed toward stimulation of regeneration through releasing of cytokines and growth factors. This may explain the appearance of mononuclear cellular infiltration in sodium selenite or taurine pretreated groups. There was prominent amelioration in the histological structure in the renal cortex in the groups exposed to mercury and pretreated with sodium selenite or taurine. Deepmala et al.89 stated that dimethyl mercury has induced tissue damage due to oxidative stress and generation of ROS and they added that the combination therapy of mercury along with zinc and selenium can prevent the tissues against mercury intoxication.

Farina et al.103 demonstrated that mercury concentration in mouse kidney decreased markedly after simultaneous administration of HgCl₂ and sodium selenite, when compared with the animals received HgCl₂ alone. They explained the protective mechanism of selenium by that mercury and selenium interact in the body of mammals to form a stable complex, thus preventing the generation of ROS. They added that the co-administration of both mercury and selenium reduces the toxicity of each other.

It has been shown that taurine has a protective effect in several tissues104 and serves as antioxidant agent.27 Erdem et al.59 proposed that the protective role of taurine occurs predominantly on the membranes of subcellular organelles, such as lysosomes and mitochondria as well as on the cytoplasmic membrane.

In conclusion, our results showed that selenium and the sulfonic amino acid taurine could protect the rat kidney from the extensive damage caused by intraperitoneal administration of mercuric chloride. The protective role of selenium and taurine may be related to their well-known ability to act as antioxidants and scavengers for ROS as well as their anti-inflammatory activity. Thus, they could be considered as potential nephroprotective agents against renal diseases.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.