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Ameliorative effect of alpha lipoic acid on CCl4 induced kidney damage of male albino rats

  • The present study is established to determine the possible protective effect of alpha lipoic acid on carbon tetra chloride (CCl4) induced renal injuries in rats. The study was conducted on 30 male rats divided into five equal groups observing the development and extent of renal injuries by histopathological and biochemical changes. Results obtained from the present study showed that CCl4 treatment can cause a significant increase in plasma urea, creatinine and MDA level and a significant reduction in antioxidant enzyme activities like SOD, Catalase in plasma and kidney tissues. Oral coadministration of alpha lipoic acid at three different doses attenuated the adverse changes. From histopathological studies it had been observed that the kidney tissue section of the untreated control group showed normal histoarchitecture, and the CCl4 treated showed severe degeneration of kidney cells. Oral coadministration of alpha lipoic acid showed comparatively well-organized glomerulus and tubules than the other lower dose group. It has been concluded that alpha lipoic acid in high doses like (100 and 200 mg/kg bodyweight/ day) had been shown the same protective effect against CCl4 induced renal damage in rats than the other lower dose group (50 mg/kg bodyweight) due to its strong anti-oxidative properties.
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Ameliorative effect of alpha lipoic acid on CCl4 induced kidney damage of male albino rats

    Corresponding author: Dilip Kumar Nandi, dilipnandi2004@yahoo.co.in
  • 1. Department of Nutrition, Physiology & Microbiology, Raja N. L. Khan Women’s College, Midnapore, West Bengal 721102, India
  • 2. Department of Physics, Jadavpur University, Kolkata, West Bengal 邮编, India
  • 3. Department of Biochemistry, Midnapore Medical College & Hospital, Midnapore, West Bengal 邮编, India

Abstract: The present study is established to determine the possible protective effect of alpha lipoic acid on carbon tetra chloride (CCl4) induced renal injuries in rats. The study was conducted on 30 male rats divided into five equal groups observing the development and extent of renal injuries by histopathological and biochemical changes. Results obtained from the present study showed that CCl4 treatment can cause a significant increase in plasma urea, creatinine and MDA level and a significant reduction in antioxidant enzyme activities like SOD, Catalase in plasma and kidney tissues. Oral coadministration of alpha lipoic acid at three different doses attenuated the adverse changes. From histopathological studies it had been observed that the kidney tissue section of the untreated control group showed normal histoarchitecture, and the CCl4 treated showed severe degeneration of kidney cells. Oral coadministration of alpha lipoic acid showed comparatively well-organized glomerulus and tubules than the other lower dose group. It has been concluded that alpha lipoic acid in high doses like (100 and 200 mg/kg bodyweight/ day) had been shown the same protective effect against CCl4 induced renal damage in rats than the other lower dose group (50 mg/kg bodyweight) due to its strong anti-oxidative properties.

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    Introduction
    • Kidney disease is the leading cause of death out of all chronic diseases in India, also worldwide[1]. Carbon tetra chloride (CCl4) is an environmental and industrial toxic pollutant with a strong nephrotoxin which is used to induce renal injury and oxidative stress in experimental animal models[23]. The toxicity of CCl4 is due to the formation of trichloromethyl radical (CCl3), which in the presence of oxygen reacts with it to form the more toxic trichloromethyl peroxyl radical (CCl3O2·) by cytochrome P-450E1 isoenzymes[4]. Lipid per oxidation reaction due to free radical generation is considered to be one of the major causes of cell membrane damage, depletion of antioxidant status and DNA injuries in the kidneys of rats[5]. Alpha-lipoic acid (ALA) is a naturally occurring dithiol compound found in different plant products like broccoli, spinach, tomato etc. that plays a fundamental role in metabolism. It has been shown to affect cellular processes, alter redox status of cells, and interact with thiols and other antioxidants[6]. ALA is a unique antioxidant because it has beneficial effects on energy production and its metabolites are capable of scavenging a variety of reactive oxygen species such as peroxynitrite[7]. Lipoic acid is used in clinical trials like accelerated chronic wound healing[8], reduced levels of asymmetric dimethylarginine (ADMA) in diabetic end-stage urinal disease patients on hemodialysis[9], attenuated acetaminophen induced uremia[10], preventing or slowing the progression of Alzheimer’s sickness[11], and reducing hypercholesterolemia and inflammation[1213]. The present study is designed to evaluate the protective role of alpha lipoic acid on CCl4 induced renal injuries in rats.

    Material and methods

      Reagents

    • Concerned different biochemical diagnostic kits like Urea, creatinine supplied by Merck (Mumbai, India). Plasma Urea and creatinine were measured using Semiautoanalyser (Merck, Micro laboratory 150). Antioxidant enzyme profiles like SOD, CAT and MDA were measured by the UV-VIS Spectrophotometer (Systronics, India). Alpha lipoic acid was purchased from Sun Pharmaceuticals-Symbiosis. All chemicals used for Biochemical tests including CCl4, Pyrogallol, etc., were purchased from Merck (Mumbai, India), HiMedia Laboratories (Mumbai, India) or Crest Biosystems (Goa, India).

    • Drug administration method

    • Carbon tetra chloride (CCl4) was injected intraperitoneally at a dose of 175 mg/mL/kg body wt. /day for 10 days[14]. Alpha lipoic acid tablets (Sun Pharmaceuticals-Symbiosis) was co-administered orally at conc. of 50, 100 and 200 mg/kg bodyweight/day for 20 days of the experiment[12].

    • Experimental subjects

      Acute toxicity study by limit test
    • An acute toxicity study of alpha lipoic acid was conducted using an acute toxic class method as per Organization of Economic Co-operation and Development (OECD) guidelines 423[15] where the limit dose of 2,000 mg kgG1 bodyweight was used. Healthy Wistar strain rats (n=5) of either sex selected by a random sampling technique were employed in this study. Wellness parameters of animals were made and recorded systematically 0.5, 4, 24 and 48 hours after dose administration for skin and fur, eyes, mucus membrane, behavioral pattern changes, tremor, convulsions, salivations, diarrhea, lethargy, sleep and mortality.

    • Main test
    • The main test was conducted on 30 healthy male albino rats (n=30). Here, a dose progression factor of two times was chosen per OECD guidelines 423[15]. The alpha lipoic acid doses selected were 50, 100 and 200 mg/kg b.wt., for 20 days continuously (OECD 2001).

      The present experiment was conducted on 30 male Wistar rats (weight of 100±15 g, supplied from Ghosh animal, animal foods and animal cages supplied by Kolkata 54). They were housed at laboratory condition for two weeks prior to experiment. Animals were housed three rats to a cage in a temperature/humidity-controlled room [(22±2) ℃] with 12-12 hours dark-light cycles. Animal care was provided according to the Guiding Principle for the Care and Use of Animals[16]. Our Institutional Animal Ethical Committee (IAEC) approved this study[17].

      The rats were randomly divided into five groups of six rats each. (1). Untreated control group (UC). (2) Untreated CCl4 Group (UCL) – rats were randomly placed in a cage with a normal diet and injected with CCl4 in a dose of 175 mg/2 mL/kg bodyweight/day for the first 10 days. (3) Treated CCl4 group and fed with alpha lipoic acid at a lower dose (TCL 50) – rats were treated similarly as UCL group and co-administered with ALA at the dose of 50 mg/0.5 mL ddH2O/kg bodyweight/day for 20 days of the experiment. (4) Treated CCl4 group with alpha lipoic acid at medium dose (TCL 100) – rats were treated as UCL group and co-administered with ALA at the dose of 100 mg/0.5 mL de-ionized water/kg bodyweight/day for 20 days of experiment. (5) Treated CCl4 group with alpha lipoic acid at high dose (TCL 200) – rats were treated as UCL group and co-administered with ALA at the dose of 200 mg/0.5 mL ddH2O/kg bodyweight/rat for 20 days of experiment. (6) At the 21th day of the experimental period, the rats were sacrificed and blood was collected from the aorta. Both kidneys were removed for histopathological analysis and different biochemical parameters.

      Biochemical estimation of blood urea. The collected blood was centrifuged to separate plasma fraction for measuring the urea level using the commercial standard Blood Urea Kit (Merck, Japan) for photometric determination of urea according to the Urease GLDH method (kinetic UV test)[18].

      Biochemical estimation of blood creatinine. The collected blood was centrifuged and plasma fraction was separated. The plasma creatinine level was measured by the commercially available standard Blood Urea Kit (Merck, Japan) by Semiautoanalyzer (Merck, Japan) using the standard protocol for photometric determination of creatinine based on Jaffe kinetic method without de-proteinization[18].

      Biochemical assay of superoxide dismutase (SOD). The kidneys were homogenized in ice-cold 100 mmol/L Tris-cocodylate buffer to give a tissue concentration of 50 mg/mL and centrifuged at 10,000 g for 20 minutes at 4 °C. The SOD activity of the supernatant was estimated by measuring the percentage of inhibition of the pyrogallol autoxidation by SOD. The buffer was 50 mmol/L Tris (pH-8.2) containing 50 mmol/L cocodylic acid (pH-8.2), 1 mmol/L EDTA and 10 mmol/L HCl. In a spectrophotometric cuvette, 2 mL of buffer, 100 lL of 2 mmol/L pyrogallol and 10 lL of supernatant were poured and the absorbance was noted in spectrophotometer at 420 nm for 3 minutes. One unit of SOD was defined as the enzyme activity that inhibited the autoxidation of pyrogallol by 50%[19].

      Biochemical assay of catalase activity (CAT). The collected blood was centrifuged and the plasma fraction was separated. The kidneys were homogenized in 0.05 mol/L Tris-HCl, pH-7.0 at 50 mg tissue/mL followed by centrifugation at 10,000 g at 4 °C for 10 minutes. Then the kidney supernatant was mixed with 0.5 mL of H2O2 and 2.5 mL of distilled water and the absorbance was spectrophotometrically measured at 240 nm. Forty microliters of tissue supernatant and plasma were added separately and six subsequent readings were noted at 30 seconds intervals[20].

      Biochemical assay of MDA in plasma and kidney tissue. The kidneys were homogenized with ice-cold phosphate buffer (0.1 mol/L, pH=7.4) at 50 mg/mL. The homogenates and blood samples were centrifuged at 10,000 g at 4 °C for 5 minutes. Then 0.5 mL supernatant and plasma were mixed separately with 0.5 mL normal saline and 2 mL of TBA-TCA mixture (0.392 g of TBA in 75 mL of 0.25 N HCl with 15 g of TCA, make up 100 ml with ethanol) and then boiled at 100 ℃ for 10 minutes. The mixture was then cooled and centrifuged at 4,000 g for 10 minutes. The whole supernatant and plasma were spectrophotometrically measured at 535 nm. Calibration was performed by using the acid hydrolysis of 1, 1, 3, 3 tetra-methoxy propane, as a standard. The MDA present within the sample was calculated by using the extinction coefficient of 1.56·105 M/cm and expressed as the unit of nM/mg of tissue or nM/mL of plasma[21].

    • Histopathological analysis of kidney tissue
    • Kidney tissues from the experimental rats were fixed in 10% buffered formalin solution embedded in paraffin wax and 5 µ sections were prepared with a rotary microtome. These thin sections were stained with hematoxylin and eosin (H and E), mounted on glass slides and observed for pathological changes under a binocular microscope according to Mani, 2010[22].

    • Statistical analysis

    • Data were expressed as mean±SE (n=6). ANOVA followed by Bonferroni multiple two-tail t-test to detect inter group differences and bars with different superscripts (*, #, @) differ from each other significantly (P<0.05)[23].

    Results

      Acute toxicity study by limit test

    • According to OECD guidelines 423, the limit test dose of 2,000 mg/kg b.wt., was given. No toxicity signs and mortality were found within 24 hours at the dose of 2,000 mg/kg b.wt., therefore, the LD50 of alpha lipoic acid is higher than 2,000 mg/kg b.wt. Next, the main test was done on three doses according to OECD guidelines 423.

    • Effect of alpha lipoic acid on plasma urea and creatinine level

    • Rats who received CCl4 resulted in a significant increase in plasma urea and creatinine levels compared to the untreated control group (P<0.05). Oral coadministration of alpha lipoic acid at three different doses (50, 100 and 200 mg/kg body wt./day) resulted in a significant decrease in plasma urea and creatinine levels compared to the CCl4 treated group (P<0.05). The decreased level of plasma urea and creatinine were more significant in the alpha lipoic acid treated higher dosage group (100 and 200 mg/kg bodyweight /day) compared to the other lower dose group (P<0.05) (Fig. 1 and 2).

      Figure 1.  Represents the effect of alpha lipoic acid coadministration at different doses on plasma urea level on CCl4 induced nephrotoxic rats.

      Figure 2.  Represents the effect of alpha lipoic acid coadministration at different doses on plasma creatinine level on CCl4 induced nephrotoxic rats.

    • Effect of alpha lipoic acid on plasma and kidney MDA level

    • Rats who received CCl4 showed a significant increase in plasma and kidney MDA levels compared to the untreated control group (P<0.05). Oral coadministration of alpha lipoic acid at three different doses (50, 100 and 200 mg/kg body wt./day) showed a significant decrease in plasma and kidney MDA levels compared to the CCl4 treated group (P<0.05). The decreased level of plasma and kidney MDA levelswas more significant in the alpha lipoic acid treated higher dosage group (100 and 200 mg/kg bodyweight/day) compared to the other lower dose group (P<0.05) and these values are resettled close to the untreated control group (Fig. 3).

      Figure 3.  Represents the effect of alpha lipoic acid coadministration at different doses on MDA level in plasma and kidney tissue on CCl4 induced nephrotoxic rats.

    • 3.4..   Effect of alpha lipoic acid on antioxidative enzyme profiles on plasma and kidney

    • Rats who received CCl4resulted in a significant decrease in SOD and catalase activities on plasma and kidney compared to the untreated control group (P<0.05). Oral coadministration of alpha lipoic acid at three different doses (50, 100 and 200 mg/kg bodyweight/day) exhibited a significant increase in SOD and catalase activities on plasma and kidney compared to the CCl4 treated group (P<0.05).The increased level in SOD and catalase activities on plasma and kidney are more significant in alpha lipoic acid treated higher dose group (100 and 200 mg/kg bodyweight/day) compared to another lower dose group (TCL 50) (P<0.05) (Fig. 4 and 5).

      Figure 4.  The effect of alpha lipoic acid co administration at different doses on catalase activities in plasma and kidney tissue on CCl4 induced kidney failure rats.

      Figure 5.  The effect of alpha lipoic acid coadministration at different doses on SOD activities in plasma and kidney on CCl4 induced nephrotoxic rats.

    • 3.5..   Histopathological findings

    • Histological examinations of the kidney section of the untreated control group have a normal morphology with a normal appearance of the glomeruli, renal tubules (Fig. 6A). Administration of CCl4 caused significant histological damage to the kidney section considering necrosed renal tubules, injured glomerulus with rupture Bowman’s capsule (Fig. 6B). Sections of kidney tissues of TCL 50 rats showed glomerulus with little loss of surrounding Bowman’s capsule and mild diffuse tubular epithelial cells (Fig. 6C). But the kidney tissue of groups TCL 100 and TCL 200 (Fig. 6D and E) represented a comparatively well-organized configuration in the renal cells structure than the TCL50 group.

      Figure 6.  The effect of alpha lipoic acid coadministration at different doses on CCl4 induced nephrotoxic rats with HE staining.

    Discussion
    • The presence of abnormally high levels of urea and creatinine in plasma are possible indicators of hepatic and kidney injuries induced through CCl4 treatment[24]. From different studies it had been reported that histopathological alteration of kidney tissue on CCl4 treated rats characterized by tubular epithelial cell alterations like atrophy, detachment of epithelial cells and tubular necrosis that ultimately leads to alteration of tubular reabsorption, decreased glomerular filtration rate (GFR) and functional overloading of nephrons with subsequent renal dysfunction resulting in abnormal renal function biomarkers[25]. The present study showed that there was a significant increase of plasma levels of urea and creatinine in CCl4 treated rats compared to the untreated control group (P<0.05). Alpha lipoic acid is effective in normalizing the antioxidant level as well as levels of creatinine and blood urea nitrogen in acetaminophen induced renal damage in rats[10,26]. Coadministration of ALA at three different doses (50, 100 and 200 mg/kg bodyweight/day from 1st day to 20th day) to CCl4-treated rats however caused a significant reduction in the plasma level of urea and creatinine thereby reversing the biochemical alterations toward normal values. In my present study it had been found that ALA coadministration at higher doses (100 and 200 mg/kg bodyweight/day) for 20 days showed significant improvement in lowering the plasma urea and creatinine levelscompared to the rats in the TCL 50 group and these values were resettled close to the normal value. CCl4 is extensively metabolized in the kidney generating extra reactive metabolites like trichloromethyl radical and its highly reactive derivative, trichloromethyl peroxyl radical (CCl3COO) by cytochrome P450E1 isoenzymes which is one of the postulated mechanisms in the pathogenesis of CCl4 nephrotoxicity[27]. It has been shown that initiated lipid per oxidation leading to accumulation of lipid per oxidation products is the source of renal and hepatic injuries[28]. The increments in lipid per oxidation with CCl4 treatment may be the consequence of increased production in free radicals and as a result the MDA level is elevated[29]. In our present study it was noted that the lipid per oxidation marker, as MDA in both plasma and kidney tissues,was significantly greater in the CCl4 treated group than the untreated control group. It has been revealed that alpha lipoic acid has been found to protect the liver and kidney against CCl4 toxicity by the reduction in the concentration of the lipid per oxidation marker, MDA[10,30]. These increased levels of MDA,in both the plasma and kidney tissues, were brought back to anormal level on oral coadministration of ALA to the CCl4 treated group at three different doses (50, 100 and 200 mg/kg bodyweight/day) by inhibiting lipid per oxidation reaction compared to the UCL group. The highest reducing activity of lipid per oxidation reaction was noted in the TCL 100 and 200 groups compared with the TCL 50 and the values were resettled close to the untreated control group. Free radical induced lipid per oxidation is considered to be one of the major causes of cell membrane damage, depletion of anti-oxidant status, and DNA injuries in kidneys of rats. The susceptibility of the liver and kidney cells to CCl4 induced oxidative insult resulted to failure of the antioxidant defense mechanism to prevent excessive free radical damage due to decreased levels of antioxidant enzymes like SOD and CAT[31]. From the present study it had been observed that SOD and CAT activities in plasma and kidney tissues were significantly depleted in the CCl4 treated group compared with the untreated control group (P<0.05). From different investigations, alpha lipoic acid showed antioxidant properties due to its lipophilic and hydrophilic properties and quenches free radicals, regenerates other antioxidants and facilitates the excretion of heavy metals[32]. Different scientists explored that ALA administration promoting an increased production of endogenous antioxidants like SOD and CAT and protected CCl4 induced liver and kidney damage in rats[10,14,33]. Oral coadministration of alpha lipoic acid at three different doses (50, 100 and 200 mg/kg bodyweight/day) group protected antioxidant machineries of the kidney as revealed from significant increases in SOD and CAT activities compared to the UCL group. Alpha lipoic acid at high doses (TCL 100 and 200) can significantly increase SOD and CAT activities in plasma as well as kidney tissue than the UC and TCL 50. After evaluation of the kidney tissue sections of the untreated control group, we found therewere no abnormal histopathological findings observed under the microscope (Fig. 6A). This renal tissue section showed normal glomerulus with intact Bowman’s capsule and normal renal tubules with tubular epithelial cells. However, the CCl4 treatment produced significant adverse morphological changes with a sloughing of the renal glomerular structure, vacuolization and necrosis (Fig. 6B). The section of kidney tissues of the TCL 50 rats showed glomerulus with little loss of the surrounding Bowman’s capsule and mild diffuse tubular epithelial cells indicated protection of kidney tissues by alpha lipoic acid (Fig. 6C). But the kidney tissue in groups TCL 100 and TCL 200 (Fig. 6D and E) represented a comparatively well-organized configuration in the renal cells structure and maximum repairment of the glomerulus and renal tubules as opposed to theTCL50 group.

      In conclusion, the results of this present study revealed that alpha lipoic acid at two higher doses (100 and 200 mg/kg bodyweight/day) alleviates the CCl4 induced renal injuries, so we have selected the lower effective dose of ALA (100 mg/kg bodyweight/day) for the protection of kidney injuries. The protective effect of alpha lipoic acid is performed through multiple ways as it scavenges free radicals generated by CCl4 and increases the activity of the antioxidant defense system by elevating the levels of antioxidant enzymes like SOD, CAT and protecting the kidney through anticipated oxidative stress. Therefore, it may be conferred that alpha lipoic acid possesses strong antioxidative properties and ameliorates renal damage caused by CCl4.

    Acknowledgment
    • The authors are grateful to the Department of Science and Technology (DST), Government of India for providing funds from the INSPIRE Fellowship (IF130382) to the first author of this work and also the CPE and BSR funds provided by the UGC, Government of India.

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