| Abstract|| |
Diazinon (DZN) is an organophosphate pesticide that is commonly used in agriculture worldwide, including in Iran, and unfortunately, it leads to a variety of negative effects on the environment, animals, and humans. Alpha-lipoic acid (ALA) is an antioxidant agent that acts via scavenging of oxygen-free radicals. Collagen IV is a component of the main base membrane structure and DZN may also affect the expression of this key protein. The aim of this study was to evaluate antioxidant properties of ALA on the expression of collagen IV, renal function, and oxidative stress induced by DZN in renal tissue. In this experimental study, 30 adult male Wistar rats were randomly divided into five groups (n = 6) including: the control group, DZN (40 mg/kg) group, ALA (100 mg/kg) group, ALA (100 mg/kg) + DZN (40 mg/kg) group, and sham group. On day 0 and after 6 weeks, the urine and blood samples were collected to measure glomerular filtration rate (GFR). After 6 weeks, the rats were anesthetized and the left kidney was used for immunohistochemistry study and the right kidney was used to evaluate the oxidative stress parameters. The results have shown that ALA significantly improved the biochemical parameters including superoxide dismutase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, and GFR. In addition, ALA significantly prevented the expression of collagen IV that was changed by DZN administration in rats. We concluded that when exposed to DZN, depletion of antioxidant enzymes is accompanied by the induction of oxidative stress that might be beneficial in monitoring DZN toxicity and alpha-lipoic acid, as an antioxidant can overcome the toxicity induced by DZN in the kidney.
|How to cite this article:|
Delavar A, Nikravesh MR, Jalali M, Valokola MG, Anbarkeh FR. The protective effect of alpha-lipoic acid on the expression of collagen IV, renal function, and oxidative stress induced by diazinon in the renal parenchyma of rat. Saudi J Kidney Dis Transpl 2020;31:1310-9
|How to cite this URL:|
Delavar A, Nikravesh MR, Jalali M, Valokola MG, Anbarkeh FR. The protective effect of alpha-lipoic acid on the expression of collagen IV, renal function, and oxidative stress induced by diazinon in the renal parenchyma of rat. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2021 Feb 28];31:1310-9. Available from: https://www.sjkdt.org/text.asp?2020/31/6/1310/308340
| Introduction|| |
Organophosphate compounds are highly toxic chemical pesticides used to control agricultural pests. These compounds are widely used because of their short life span in the environment. Diazinon (DZN) is a typical organo-phosphorus (OP) pesticide that is widely used by different people, such as farmers and orchard owners in different regions of Iran because they believe that these compounds are not problematic. However, various studies on the adverse effects of DZN suggest that this chemical composition has a wide range of harmful biochemical effects even in nonlethal doses. Studies have shown that DZN leads to reduced weight in the reproductive organs, increased abnormalities, and death of sperm, and it reacts with macromolecules and micro-molecules, resulting in genetic and cellular damage. In addition, oxidative damage caused by DZN toxicity leads to hematologic disorders, cardiac toxicity, liver toxicity, and nephrotoxicity and neurological disorders. The severity of the effect of DZN depends on the dose and how it is absorbed in the body. DZN is mainly diffused through the skin, eyes, respiration, and swallowing. The main mechanism of these toxic compounds is due to the similarity of its structure with cholinesterase substrate, namely acetylcholine. This enzyme is inhibiting and cannot clean the synaptic cleft of acetylcholine, and as a result, message transmission is disrupted. Some researchers consider the increase in lipid peroxidation and the production of free radicals derived from the metabolism of organophosphate pesticides as the main mechanism of cell destruction. Considering the harmful effects of this poison and its widespread use in agriculture, various ways to deal with its destructive effects have been considered, including the use of anti-oxidants even at a low concentration, which inhibit the oxidation of oxidizing materials and protect the cell against the adverse effects of environmental factors. In the present study, alpha-lipoic acid (ALA) or thioctic acid was used, which is a cofactor for the mitochondrial dehydrogenase enzyme that is in metabolism and energy production. On the one hand, this material can directly destroy the proxyl free radicals produced in the blue phase and the microsomal membrane. On the other hand, by resuscitating ascorbyl and chromenoxyl, it increases the strength of other antioxidants (Vitamins E and C). Moreover, other studies have shown that alpha-lipoic acid can be effective in treating several models of oxidative stress such as ischemia/reperfusion, diabetes, and cataract formation. Since the renal injury caused by DZN is attributed to oxidative stress and given that the kidneys are one of the main centers of waste material removal, drugs, and poisons in the body, and compared to other organs of the body, such as the brain and the liver, remaining toxins are higher in the kidneys, which can cause further damage to the renal parenchymal. One of the proteins that make up kidney parenchyma is collagen IV, wherein the subunits of α1 (IV) to α6 (IV) are formed, consisting of three primary spirals or promoters, each of which is considered as a tropocollagen to form this polymer. In addition, in the manufacturing of this spiral, various compounds comprise six subunits of type IV collagen, each of which is expressed by an independent gene. Therefore, any mutation leads to a defect in the synthesis of this type of collagen, and ultimately, it can lead to impaired glomerular function. Therefore, the aim of this study was to evaluate the effect of DZN and the protective effects of ALA on oxidative stress and collagen IV expression in the renal parenchymal of rats.
| Materials and Methods|| |
Animals and treatment
In this experimental study, 30 adult male Wistar rats (age: 2 months old), with a weight range of 220–250 g, were randomly divided into five groups (n = 6): control group (no intervention); DZN group, 40 mg/kg (Karon Co., Ltd., China); ALA group, 100 mg/kg (Sigma-Aldrich, USA), co-treatment group (DZN 40 mg/kg and ALA 100 mg/kg); and sham group that received ethanol at the same volume of the DZN group. In this study, DZN (diluted in ethanol) and ALA were administered orally. On day 0 and after 6 weeks, the rats were placed in a metabolic cage for 24 h and urine and orbital blood samples were collected to evaluate biochemical parameters. Finally, the rats were anesthetized and were humanely killed. Then, the left kidney was used for immunohistochemistry study and the right kidney was used to measurement oxidative stress factors. The rats were kept in standard conditions (at 23°C ± 2°C and 12-h light/dark cycle) and they were allowed free access to food and water during the experiment. All the experimental protocols were approved by the Ethics Committee of the Mashhad University of Medical Science (IR.MUMS.fm.REC.1396.836).
For immunohistochemical detection of collagen IV, serial sections (5 μm) were mounted on poly-L-lysine slides. Sections were deparaffinized with xylene and rehydrated through descending concentrations of ethanol. Enzymatic antigen retrieval was carried out with trypsin (0.05%) in PBS. Sections were preincubated in 0.025% Triton X-100 in PBS at room temperature. Subsequently, the specimens were intubated with 3% H2O2 to blocking nonspecific interactions and placed in 5% goat serum. Then, sections were reacted with primary antibody (anticollagen IV, Abcam, Cambridge, USA) diluted 1:400 in PBS with 1% bovine serum albumin (BSA) overnight (for 24 h at 4°C). The next day, the sections were washed with 0.025% Triton X-100 in PBS at room temperature. Then, the specimens were incubated with goat polyclonal secondary antibody (Abcam, Cambridge, USA) and diluted 1:800 in PBS with 1% BSA for 2 h. Afterward, the sections were washed for 10 min in PBS and finally reacted with 0.03% solution of 3, 3-diaminobenzidine containing 0.3% H2O2. To create a background color, specimens were stained with hematoxylin. Finally, the sections were dehydrated in ethanol, cleared in xylene, and mounted in glass slides. Collagen IV optical density of tubules and renal glomeruli in different groups was done using ImageJ software and this formula:
The maximum intensity in red–green–blue images was 255 and the mean intensity was calculated based on the ImageJ software. The numbers obtained from this software were analyzed by statistical tests.
Determination of glomerular filtration rate
Glomerular filtration rate (GFR) was estimated using creatinine clearance (CrCl). Blood and urine (24 h urine) samples were taken from all rats before the treatment (day 0) and after the treatment (day 42) to determine the serum and urine Cr levels, respectively, by the Cr kit (Pars Azmoon Company, Tehran, Iran). The GFR was calculated using the following equation:
GFR (mL/min) = CrCl =[urine Cr (mg/dL) × urine output (mL/min) ÷ plasma Cr (mg/dL)]
Determination of glutathione peroxidase activity
Glutathione peroxidase (GPx) activity was evaluated according to the technique of Mohandas, which registers the vanishing of nicotinamide adenine dinucleotide phosphate (NADPH) by its absorbance at 340 nm.
Determination of glutathione reductase activity
Glutathione reductase (GR) activity in the kidney was evaluated through the method of Carlberg. Enzyme activity was determined by measuring the vanishing of NADPH at 340 nm by a spectrophotometer (model 4001/4) and was assessed as nmol NADPH oxidized/ min mg tissue-1 using the molar extinction coefficient of 6.22 × 103 Mcm-1.
Determination of glutathione S-transferase activity
Glutathione S-transferase (GST) activity was determined using the method of Habig. The changes in absorbance were registered at 340 nm and enzyme activity was evaluated as nmol CDNB conjugate formed/min mg tissue-1 using a molar extinction coefficient of 9.6 × 103 M-1 cm-1.
Determination of superoxide dismutase enzyme activity
The superoxide dismutase enzyme activity (SOD) activity was evaluated according to the method explained by Sun. SOD activity was measured at 560 nm at 37°C and was calculated using the inhibition percentage of the formazan formation. The SOD enzyme activity is expressed as unit/mg tissue so that one unit of SOD is descripted as the amount of sample which led to 50% inhibition of NBT reduction.
| Statistical Analysis|| |
The statistical analysis was performed by Statistical Package for the Social Sciences (SPSS) software for Windows version 16.0 (SPSS Inc., Chicago Illinois, USA). Normality test (Kolmogorov–Smirnov) was done. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc comparison test. All data are presented as mean ± SEM. P <0.05 was regarded statistically significant.
| Results|| |
Effect of diazinon and alpha-lipoic acid on the collagen IV expression in the glomerular and tubular basement membrane of the kidney
According to the optical density provided by the ImageJ software, one of the most important findings of this study was that DZN significantly increased the expression of collagen IV in the glomerular and tubular basement membrane kidney compared to the control group (P <0.05). Furthermore, ALA in the co-treatment group significantly decreased the synthesis of collagen IV in the glomerular and tubular basement membrane compared to the DZN group (P <0.05) [Figure 1]f and [Figure 2]f). Moreover, in the DZN group, the collagen density significantly enhanced the base membrane, and this intensity of collagen expression was lower in other groups [Figure 1] and [Figure 2].
|Figure 1: Photomicrographs of renal glomeruli after reaction to anticollagen IV. Control (A), DZN (B), ALA + DZN (C), ALA (D) and sham (E) groups. The locations of collagen IV are brown (arrows). (F) Optical density of collagen IV in renal glomeruli. All data are presented as mean ± SEM.|
*P <0.05 compared to the control group and #P < 0.01 compared to DZN group. OD: Optical Density, ALA: Alpha lipoic acid, DZN: Diazinon.
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|Figure 2: Transverse section of renal tubules after IHC reaction. Control (A), DZN (B), ALA + DZN (C), ALA (D), and sham (E) groups. In these photomicrographs, reaction to anticollagen IV is observed in brown color (arrows). (F) Optical density of collagen IV in renal tissue tubules. All data are presented as mean ± SEM.|
*P <0.05 compared to the control group, #P <0.01 compared to DZN group. OD: Optical Density, ALA: Alpha lipoic acid, DZN: Diazinon.
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Effect of diazinon and alpha-lipoic acid on the glomerular filtration rate
The mean ± SEM of GFR difference is shown in [Table 1]. Comparison of GFR difference results between days 0 and 42 showed that in the DZN group, the amount of GFR was decreased compared to the control and other groups (P <0.05). The GFR value in the DZN+ALA and ALA groups on day 42 increased (P <0.05).
|Table 1: Glomerular filtration rate difference (mL/min) in all experimental groups (n = 6).|
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Protective effect of alpha-lipoic acid on the glutathione peroxidase and glutathione reductase activity in renal tissue following exposure to diazinon
In this study, DZN significantly reduced GR and GPX levels of kidney in compared to the control group (P <0.05). In addition, ALA in the co-treatment (ALA + DZN) group and ALA group increased the GR and GPX levels compared to the DZN group (P <0.05) [Figure 3]a and [Figure 3]b.
|Figure 3: Effect of diazinon and alpha lipoic acid on glutathione peroxidase (A) and glutathione reductase activity (B). All data are presented as mean ± SEM.|
*P <0.05 compared to control, #P <0.05 compared to DZN group.
DZN: Diazinon, ALA: Alpha lipoic acid.
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Protective effect of alpha-lipoic acid on glutathione S-transferase and superoxide dismutase activity in renal tissue following exposure to diazinon
The result of this study showed that DZN significantly increased the mean ± SEM of GST and SOD level in comparison to the control group in the kidney tissue (P <0.05) [Figure 4]a and [Figure 4]b. Furthermore, the ALA-treatment group significantly decreased GST activity compared to the DZN group (P <0.01) [Figure 4]A.
|Figure 4: Effect of diazinon and alpha-lipoic acid on glutathione S-transferase (A) and superoxide dismutase activity (B). All data are presented as mean ± SEM.|
*P <0.05 compared to control group, ##P <0.01 compared to DZN group.
DZN: Diazinon, ALA: Alpha-lipoic acid.
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| Discussion|| |
Diazinon as an organophosphate pesticide is able to produce free radicals and disturbance in the antioxidant systems in the body. The kidney is an essential organ of the body that performs important functions, including maintaining homeostasis, the regulation of the extracellular environment, such as detoxification and the elimination of toxic metabolites and medications. Therefore, the kidney can be considered as a target organ for poisons. DZN and its metabolites can be associated with damage to the kidney and kidney tubules. This study shows that DZN causes oxidative stress and renal dysfunction and increases the synthesis of glomerular basement membrane collagen and renal tubules. It is believed that the use of natural and synthetic antioxidants can be useful in the treatment of nephro-toxicity. The observed protective effects can be related to the antioxidant properties of ALA. The results of this study show that DZN leads to the destruction of the enzymatic and nonenzymatic antioxidant chain, including GR, GST, GPx, and SOD in the kidney. GR preserves glutathione in a reduced form. Therefore, reduction of GR activity after DZN administration may be due to decreased glutathione and the reduced availability of NADPH. GPx is an enzyme that reduces the amount of hydrogen peroxide to protect cells from oxidative damage. Reducing GPx levels after exposure to DZN may result in peroxide accumulation. The activity of these enzymes can be one of the factors influencing the amount of glutathione in the tissue because DZN administration reduces glutathione and inhibits antioxidant enzymes, including the enzymes involved in glutathione metabolism. Finally, it is known that DZN catalyzes the production of reactive oxidants. The GST enzyme uses glutathione to increase the solubility of toxins and eliminates them from the body. Therefore, it plays an important role in protecting the tissues against oxidative stress. In this study, DZN at a dose of 40 mg/kg increased the activity of the GST enzyme in the kidney tissue. This increase represents an increase in glutathione consumption and an increase in the body’s defense against this toxin to neutralize it. Several studies have shown that GST activity increased after exposure to OP pesticides. Depletion of glutathione may increase lipid peroxidation, damage to DNA, and decrease resistance to oxidative damage. SODs belong to the family of antioxidant enzymes, which is essentially a protective enzyme that removes superoxide ions produced by oxidative stress. The results of this study indicate that DZN administration significantly increases the activity of SOD enzymes, and the increase in SOD activity, by decreasing the superoxide radical, causes an increase in H2O2 levels in the kidney., In the current study, ALA (100 mg/kg) reduced SOD and inhibited oxidative stress caused by DZN. Previous studies have also shown that DZN induces oxidative stress in rat kidney tissues and increases SOD. To evaluate the function of the kidneys, the GFR, which is equivalent to CrCl, has been considered. Therefore, any change in the amount of Cr leads to a change in the amount of GFR, which is the best indicator of kidney function. In a previous study, DZN was given to mice and several biochemical markers were appraised. The results showed that the amount of Cr did not change, which could be due to the low dose and the difference in how it was consumed. However, in Shah’s study, oral administration of DZN for 8 weeks showed an increase in the serum Cr level, which is consistent with our results. The present study shows that ALA with a dose of 100 mg/kg significantly increased the GFR compared to the DZN-treated group. Among the basement membrane components, collagen is one of the most abundant compounds and among them, collagen type IV creates the main structure of this sector. ROS directly damages important biological macromolecules and leads to the production of advanced protein oxidation and advanced glycation products that not only indicate oxidative stress but also damage the kidney. In this study, immunohistochemistry techniques were utilized. The intense reaction to tonality in the glomerular basement membrane and tubules in the glycosaminoglycans group confirms the increase of this type of collagen. Hence, it seems that collagen type IV, as one of the most important structural elements of the membrane, can also affect renal function. One of the causes of kidney fibrosis is inflammation and the role of macrophages in inflammation and promoting glomerular and interstitial fibrosis has been proven. Their interaction with epithelial cells in the kidney leads to interstitial fibrosis in damaged kidneys. Various studies have shown that ALA, due to its anti-inflammatory properties, is capable of inhibiting macrophage infiltration and thus leads to inhibiting the progression of kidney fibrosis. In addition, renal fibrosis has been shown to increase glomerular thickness, which indicates a decreased activity of metalloproteinase that is due to the increase in the thickness of the membrane in the glomeruli and tubules, wherein there is a direct relationship with deficiency and decreased filtration in the kidney. Therefore, our results show that these changes were reduced in the ALA group, which is according to studies that show ALA improves the expression of the values of matrix metalloproteinase in tubular epithelial cells and in some interstitial cells adjacent to damaged kidney tubules. Although the use of the immunohistochemistry study showed collagen changes, the use of other techniques, including western blot and real-time polymerase chain reaction, is recommended to confirm changes in the extracellular matrix.
| Conclusion|| |
The present study reveals a promising role for ALA to meliorate DZN-induced renal injury, possibly through its antioxidant properties. The finding suggests that ALA reduces the DZN-induced functional damage of kidneys in rats and helps improve the glomerular filtration rate.
Conflict of interest: None declared.
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Mohammad Reza Nikravesh
Department of Anatomy and Cell Biology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad
[Figure 1], [Figure 2], [Figure 3], [Figure 4]