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. 2023 May 4;6(2):168–177. doi: 10.1002/ame2.12315

Ameliorative effects of glycine on cobalt chloride‐induced hepato‐renal toxicity in rats

Oluwafikemi Temitayo Iji 1, Temitayo Olabisi Ajibade 2,✉, Oluwaseun Olanrewaju Esan 3, Omolola Victoria Awoyomi 1, Ademola Adetokunbo Oyagbemi 2, Moses Olusola Adetona 4, Temidayo Olutayo Omobowale 2, Momoh Audu Yakubu 5, Oluwafemi Omoniyi Oguntibeju 6, Evaristus Nwulia 7
PMCID: PMC10158950  PMID: 37141004

Abstract

Background

The important roles of liver and kidney in the elimination of injurious chemicals make them highly susceptible to the noxious activities of various toxicants including cobalt chloride (CoCl2). This study was designed to investigate the role of glycine in the mitigation of hepato‐renal toxicities associated with CoCl2 exposure.

Methods

Forty‐two (42) male rats were grouped as Control; (CoCl2; 300 ppm); CoCl2 + Glycine (50 mg/kg); CoCl2 + Glycine (100 mg/kg); Glycine (50 mg/kg); and Glycine (100 mg/kg). The markers of hepatic and renal damage, oxidative stress, the antioxidant defense system, histopathology, and immunohistochemical localization of neutrophil gelatinase associated lipocalin (NGAL) and renal podocin were evaluated.

Results

Glycine significantly reduced the markers of oxidative stress (malondialdehyde content and H2O2 generation), liver function tests (ALT, AST, and ALP), markers of renal function (creatinine and BUN), and decreased the expression of neutrophil gelatinase‐associated lipocalin (NGAL) and podocin compared with rats exposed to CoCl2 toxicity without glycine treatment. Histopathology lesions including patchy tubular epithelial necrosis, tubular epithelial degeneration and periglomerular inflammation in renal tissues, and severe portal hepatocellular necrosis, inflammation, and duct hyperplasia were observed in hepatic tissues of rats exposed to CoCl2 toxicity, but were mild to absent in glycine‐treated rats.

Conclusion

The results of this study clearly demonstrate protective effects of glycine against CoCl2‐induced tissue injuries and derangement of physiological activities of the hepatic and renal systems in rats. The protective effects are mediated via augmentation of total antioxidant capacity and upregulation of NGAL and podocin expression.

Keywords: cobalt chloride, hepatotoxicity, nephrotoxicity, oxidative stress, podocin

Glycine offered protection against cobalt chloride hepato‐renal toxicity. The two novel biomarkers of renal damage Neutrophil gelatinase‐associated lipocalin (NGAL) and podocin we significantly down‐regulated in the present study. We report for the first time hepato‐renal protection of glycine on cobalt chloride toxicity.

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1. INTRODUCTION

Amino acids, which are organic substances containing amine and carboxylic acid functional groups, are the basic units of protein. 1 Amino acids contribute significantly to the modulation of diverse arrays of cellular metabolic processes including biosynthesis of lipids, glutathione, nucleotides, glucosamine, and polyamines. 2 , 3 Glycine, the simplest amino acid in mammals, is involved in the biosynthesis of creatine, haeme, purines, and deoxyribonucleic acid. 4 As a precursor of glutathione, glycine exerts protective effects on mammalian tissues, and has been reported to mitigate oxidative stress‐mediated tissue injury by abrogating free radical production. 5 In hepatic tissues, glycine has been reported to abrogate the development of liver fibrosis via inhibiting activation of Kupffer cells and preventing the release of pro‐inflammatory and pro‐fibrogenic cytokines. 6 , 7 Moreover, a combination of glycine and N‐acetylcysteine has been reported to ameliorate oxidative stress, mitochondrial impairment, inflammation, and endothelial dysfunction associated with glutathione deficiency in aged mice, thus suggesting potential antioxidant and anti‐inflammatory effects of glycine in vivo. 7 As regards glycine‐mediated renoprotection, modulation of total antioxidant capacity and prevention of renal structural damage by glycine have been linked to enhancement of glutathione synthesis and suppression of renal NADPH‐oxidase expression in rats. 8

The physiological roles of cobalt (Co2+) are mostly seen in the modulation of nervous system functioning and formation of new erythrocytes. 9 Cobalt modulates the activities of vitamin B12 (cyanocobalamin), which potently modulates mammalian cell growth, development, and erythropoiesis. 10 Cobalt is a coenzyme for several cellular processes including fatty acid oxidation, deoxyribonucleic acid biosynthesis, and cellular energy production. 11 , 12 Unfortunately, exposure to cobalt in quantities beyond the physiological dose has been reported to cause severe deleterious effects in various mammalian systems including the nervous, renal, hepatic, and haemopoietic systems. 13 , 14 In humans, cobalt toxicity resulting from high levels of exposure has been associated with inhalation of cobalt dust and metal‐on‐metal total hip arthroplasty and shaving of cobalt‐chromium secondary to retained ceramic particles from a failed femoral head prosthesis, causing the release of cobalt into systemic circulation. 15 Earlier studies also reported increased aspartate amino transferase and alanine aminotransferase (markers of hepatic function) following increased cobalt administration, with severity of alteration increasing with the duration of toxic exposure. 16 Likewise, administration of CoCl2 has been demonstrated to cause a significant increase in the activity of alkaline phosphatase and total bilirubin in serum following chronic exposure. 17

Although, the potent oxidative stress‐inducing ability of CoCl2 has been well described in several organs and tissues, the role of oxidative stress in the pathogenic mechanisms associated with hepatic toxicities and associated renal dysfunctions has not been well elucidated. Moreover, the mitigation of hepato‐renal toxicity by glycine has not been reported. Therefore, this study was designed to investigate the probable role of oxidative stress in the pathogenesis of CoCl2‐mediated hepatic and renal toxicities, and the modulatory role of glycine, as a potent antioxidant, in the hepatic and renal toxicities associated with CoCl2 exposure.

2. METHODS

2.1. Experimental design

The in vivo evaluation of the modulatory role of glycine on oxidative stress‐mediated CoCl2‐induced toxicity in a murine model was carried out using forty‐two (42) adult male rats. After an acclimatization period of 2 weeks, the rats were randomly assigned into the following 6 groups consisting of 7 rats each: Control; (CoCl2; 300 ppm); CoCl2 + Glycine (50 mg/kg); CoCl2 + Glycine (100 mg/kg); Glycine (50 mg/kg); Glycine (100 mg/kg). CoCl2 was administered in drinking water (300 ppm), while glycine was administered by oral gavage to the experimental rats once daily for seven consecutive days.

2.2. Ethical regulations

The study was conducted in accordance with the provision of the University of Ibadan ACUREC with approval code (UI‐ACUREC/100–1021/9).

2.3. Chemicals

The following analytical‐grade chemicals and reagents were utilized over the course of this study: glycine (CAS No.: 56–40‐6), cobalt chloride (CAS No.: J54839), xylenol orange (CAS NO.: 3618‐43‐7), 1,2‐dichloro‐4‐nitrobenzene (CDNB) (CAS No.: 99–54‐7), 5,5′‐dithio‐bis‐2‐nitrobenzoic acid (DTNB); (CAS No.: 69–78‐3), trichloroacetic acid (TCA); (CAS No.:76–03‐9), thiobarbituric acid (TBA); (CAS No.: 504–17‐6), reduced glutathione (GSH); (CAS No.:70–18‐8), hydrogen peroxide (CAS No.:7722‐84‐1), sodium hydroxide (CAS No.: 1310‐73‐2), epinephrine (CAS No.: 51–43‐4). All were obtained from Sigma (St Louis, MO, USA).

Biotinylated secondary antibodies: 2‐step plus Poly‐HRP Anti Mouse/Rabbit IgG Detection System with DAB solution and primary antibodies against Angiotensin Converting Enzyme1 Polyclonal Antibody (E‐AB‐16159: 1:500 Dilution) and Podocin (NPHS2) Polyclonal Antibody (E‐AB‐1663: 1:150 Dilution) were purchased from Elabscience Biotechnology®, China. All other chemicals used for this study were of analytical grade.

2.4. Serum preparation

At the end of the experimental period, blood was collected in xylazine/ketamine‐anesthetized rats from the retro‐orbital venous plexus using sterile heparinized capillary tubes into plain (antioxidant free) sample bottles. The blood samples were allowed to stand at room temperature (25°C) for 30 min to allow for clotting of the whole blood and separation of serum. A sterile Pasteur pipette was used to carefully harvest the sera from the clotted blood into clean antioxidant free sample bottles that were then preserved in a refrigerator at 4°C.

2.5. Preparation of hepatic and renal post mitochondrial fractions

Under anesthesia, the liver and the kidneys were carefully excised, rinsed, weighed, and kept on ice in a cooling chamber. The tissue samples were kept in homogenizing buffer (0.1 M phosphate buffer, pH 7.4) on ice to ensure preservation of enzyme activity and subsequently homogenized with a Teflon homogenizer. Thereafter, the homogenates were centrifuged at 10 000 g for 10 min at −4°C and supernatant stored until needed for bioassay.

2.6. Biochemical analysis for antioxidant status

In this study, non‐enzymatic antioxidants such as the reduced glutathione were analysed as described by Beutler et al, 18 whereas the activities of enzymatic antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione S‐transferase (GST) were determined as described by Rotruck et al, 19 Misra and Fridovich, 20 and Habig et al, 21 respectively. Hydrogen peroxide generation was estimated as described by Wolff, 22 whereas themalondialdehyde (MDA) level was calculated as described by Varshney and Kale. 23 Lipid peroxidation in μmol MDA formed/mg protein was computed with a molar extinction coefficient of 1.56 × 105 M−1 cm−1.

2.7. Biochemical analysis for the markers of hepatic and renal functions

Aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, and blood urea nitrogen (BUN) levels were assayed in plasma using Randox kits according to the procedures highlighted by the manufacturer (Randox Laboratories Ltd, Crumlin, UK) (Table 1).

TABLE 1.

Markers of renal function in serum of rats.

Parameters Control CoCl2 CoCl2 + 50 mg/kg Glycine CoCl2 + 100 mg/kg Glycine Glycine 50 mg/kg Glycine 100 mg/kg
Creatinine (mg/dl) 0.73 ± 0.02 1.94 ± 0.03 a 1.37 ± 0.01 0.94 ± 0.02 b 0.78 ± 0.03 b 0.80 ± 0.01 b
BUN (mg/dl) 63.50 ± 4.21 142.23 ± 5.8 a 116.45 ± 3.11 78.55 ± 5.12 b 65.61 ± 3.65 b 68.24 ± 4.32 b
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Abbreviation: BUN, blood urea nitrogen.

a

Indicates significant (p < 0.05) increase compared with control.

b

Indicates significant (p < 0.05) decrease compared with CoCl2.

2.8. Urinalysis

On the last day of experiments, rats were placed in metabolic cages and 2–3 mL of fresh urine was collected in clean sample bottles for urinalysis using standard urinary assay kit (Randox Laboratories Ltd). The urine was analysed for ascorbic acid, blood, bilirubin, glucose, protein, leucocytes, and specific gravity (Table 2).

TABLE 2.

Urinalysis.

Group ASC BLD BIL KET GLU PRO LEU pH SG
Control − − − − − − − 8 1.000
CoCl2 − − − + − + 2+ 9 1.005
CoCl2 + Glycine 50 mg/kg − − − − − − 1+ 8 1.005
CoCl2 + Glycine 100 mg/kg − − − − − − 1+ 8 1.005
Glycine 50 mg/kg − − − − − − 1+ 8 1.005
Glycine 100 mg/kg 2+ − − − − − 1+ 8 1.005
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Abbreviations: ASC, ascorbic acid; BLD, blood; BIL, bilirubin; GLU, glucose; LEU, leucocytes; PRO, protein; SG, specific gravity; −, negative; +, positive.

2.9. Histopathology

Small slices of the liver and kidneys were collected in 10% buffered formalin for proper fixation. These tissues were processed and embedded in paraffin wax. Sections 5–6 μm thick were made and stained with Hematoxylin and Eosin for histopathology. 24

2.10. Immunohistochemistry

The immunohistochemical staining of podocin and NGAL was evaluated in renal tissues as described by Oyagbemi et al 25 using a 2‐step plus Poly‐HRP Anti Mouse/Rabbit IgG Detection System with DAB solution. Paraffin embedded tissue samples on slides were dewaxed in xylene solution for 2 min and afterwards hydrated in different concentrations of ethanol (100%, 90%, 80% and 70%) for 2 min each. The retrieval of antigen in dewaxed hydrated tissues was carried out prior to endogenous peroxidase blocking, and subsequent addition of goat serum to prevent nonspecific binding. The tissues were then probed with primary polyclonal antibodies for podocin and NGAL. This was followed by the addition of secondary antibody and incubation of slides in a humidifying chamber at 25°C for 2 h. After the incubation period, diaminobenzidine (DAB) was added in the dark, and the reaction was terminated with deionized water. The slides were immersed in Hematoxylin (Sigma‐Aldrich, USA) for 3 s before rinsing with PBS, and were further immersed in 80%, 90%, and 100% of ethanol, and then xylene (100%) for 2 min each. The slides were allowed to dry, and a DPX mountant was applied. Sections were observed with a light microscope (Leica LAS‐EZ®) using Leica software application suite version 3.4 equipped with a digital camera.

2.11. Statistical analysis

Data obtained were analyzed with one‐way ANOVA with Tukey post hox test at a 95% confidence limit. All values were expressed as means ± S.D. The test of significance between two groups was estimated by Student's t test.

3. RESULTS

3.1. Hepatic and renal oxidative stress indices and antioxidant defense status

Our findings revealed significant differences (p < 0.05) in the content of MDA and H2O2 levels of hepatic and renal tissues of rats exposed to CoCl2 compared to the control and rats administered only glycine at 50 mg/kg and 100 mg/kg. The levels of the markers of oxidative stress MDA and H2O2 were significantly (p < 0.05) lower in rats administered both glycine and CoCl2 compared to rats that received CoCl2 without glycine treatment (Figure 1A,B).

FIGURE 1.

FIGURE 1

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Hepatic and renal oxidative stress indices and antioxidant defense status. (A), Malondialdehyde (MDA) level in the liver and kidney of rats (B), Hydrogen peroxide (H2O2) level in the liver and kidney of rats. (C), Reduced glutathione (GSH) values in the liver and kidney of rats. (D), Glutathione peroxidase (GPx) activity in the liver and kidney of rats. (E), Superoxide dismutase (SOD) activity in the liver and kidney of rats. (F), Glutathione S‐transferase activity in the liver and kidney of rats. Superscript (a) indicates significantly increased level (p < 0.05), relative to control, but superscript (b) indicates a significant difference relative to cobalt chloride.

The level of reduced glutathione (GSH) in rats treated with glycine alone was significantly higher than the control group and CoCl2 only group. Although rats treated with glycine and CoCl2 had significantly lowered GSH levels compared with control, the levels were significantly (p < 0.05) higher than those of rats treated with CoCl2 without glycine (Figure 3). The activities of GPx, SOD, and GST were significantly higher in rats administered both CoCl2 and glycine compared to those exposed to CoCl2 alone (Figure 1C–F). Also, the activities of the antioxidant enzymes in the glycine‐treated rats were insignificantly different from those of the control rats.

FIGURE 3.

FIGURE 3

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Photomicrographs of kidney tissues. (A), Control showing no visible lesion; (B), Cobalt Chloride (CoCl2) showing patchy tubular epithelial necrosis and inflammation; (C), CoCl2 + 50 mg/kg glycine showing no visible lesion; (D), CoCl2 + 100 mg/kg glycine showing no visible lesion; (E), 50 mg/kg glycine showing no visible lesion; (F), 100 mg/kg glycine showing no visible lesion. H&E. Magnification × 400.

3.2. Hepatic and renal function tests of CoCl2 exposed rats

In this study, the activities of the analysed markers of hepatic function ALT, AST, and ALP were significantly (p < 0.05) higher in rats exposed to CoCl2 alone when compared with the control group (Figure 2). However, the activities of the enzymes were significantly (p < 0.05) lower in rats treated with glycine compared with those exposed to CoCl2 alone. Likewise, serum bilirubin and creatinine levels increased significantly (p < 0.05) in rats exposed to CoCl2 alone relative to the control and glycine‐treated groups (Figure 2).

FIGURE 2.

FIGURE 2

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Markers of hepatic function in the serum of rats. Superscript (a) indicates significantly increased level (p < 0.05), relative to control, but superscript (b) indicates significantly decreased activity relative to cobalt chloride.

3.3. Urinalysis of metabolites

The result of the urinalysis revealed the presence of ketone bodies and protein in the urine of CoCl2−exposed rats, in contrast to the urine of rats that received glycine.

3.4. Histopathology of the liver and kidney

In this study, CoCl2 intoxication induced several pathological lesions including patchy tubular epithelial necrosis, tubular epithelial degeneration, and periglomerular inflammation in the kidney tissues of rats (Figure 3). The observed lesions were not, however, visible in glycine‐treated rats. Moreover, histopathological lesions such as severe portal hepatocellular necrosis, inflammation, and duct hyperplasia were seen in the hepatic tissues of rats exposed to CoCl2 toxicity, but these lesions were mild to absent in glycine‐treated rats (Figure 4).

FIGURE 4.

FIGURE 4

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Photomicrographs of liver tissues. (A), Control showing no visible lesion; (B), Cobalt Chloride (CoCl2) showing severe portal hepatocellular necrosis, inflammation, and duct hyperplasia; (C), CoCl2 + 50 mg/kg glycine showing no visible lesion; (D), CoCl2+ 100 mg/kg glycine showing no visible lesion; (E), 50 mg/kg glycine showing no visible lesion; (F), 100 mg/kg glycine showing no visible lesion. H&E. Magnification × 400.

3.5. Immunohistochemistry of acute renal injury

The immunohistochemical evaluation of NGAL was found to be higher in renal tissues of rats exposed to the toxic effects of CoCl2 without glycine treatment relative to the control group, and rats treated with glycine following CoCl2 exposure (Figure 5). In the same vein, the expression of podocin, a protein highly expressed in acute renal injury, was found to be higher in CoCl2−exposed rats relative to the control (Figure 6). However, lower expression of podocin was observed in glycine‐treated rats when compared to the rats administered CoCl2 alone. The expression of podocin was also lowered in rats administered 100 mg/kg glycine than in rats administered 50 mg/kg glycine.

FIGURE 5.

FIGURE 5

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The immunohistochemistry of renal neutrophil gelatinase‐associated lipocalin (NGAL). Group A, Control; Group B, CoCl2; 300 ppm; Group C, CoCl2 + 50 mg/kg Glycine; Group D, CoCl2 + 100 mg/kg Glycine; Group E, 50 mg/kg Glycine; Group F, 100 mg/kg Glycine. Slides stained with high definition Heamtoxylin. Magnification × 100.

FIGURE 6.

FIGURE 6

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The immunohistochemistry of renal podocin. Group A, Control; Group, CoCl2; 300 ppm; Group C, CoCl2 + 50 mg/kg Glycine;), Group D, CoCl2 + 100 mg/kg Glycine; Group E, 50 mg/kg Glycine; Group F, 100 mg/kg Glycine. Slides stained with high definition Hemotoxylin. Magnification × 100.

4. DISCUSSION

The liver and the kidneys are important organs of detoxification and excretion. Therefore, they are important in the protection of mammals from noxious xenobiotics including drugs and other toxicants. Unfortunately, liver and kidneys are highly susceptible to toxic effects from injurious chemicals and/or toxicants following acute or chronic exposures. Although, the liver has an unparalleled ability to regenerate damaged hepatocytes, toxicant‐induced nephrotoxicity is usually irreversible and life‐threatening. In this study, the deleterious effects of CoCl2 on hepatic and renal tissues manifested as increased biomarkers of oxidative stress including H2O2 generation and MDA content, and reduction in the activities of GPx, GST, SOD, and GSH content. Our observations in this study corroborated an earlier report by Ali et al, 26 who described remarkable elevations in MDA levels accompanied by significant depletions in the GSH content and SOD activity in the brain, liver, and kidney of rats. Furthermore, there are several reports that cobalt mediates organ specific injuries in mammals and nonmammalian organisms via the induction of oxidative processes. 27 , 28

Oxidative stress is well characterized as an important mechanism in the pathophysiology and pathogenesis of liver diseases. 29 Expectedly, biomolecules with strong antioxidant activities such as melatonin and vitamin E have been demonstrated to potently mitigate oxidative stress‐mediated hepatic dysfunction. 30 , 31 In this study, the potentiation of the actions of antioxidant molecules including GSH, GPx, GST, and SOD in the liver of CoCl2‐exposed rats and those treated with glycine suggested a probable hepatoprotective effects for this amino acid as earlier reported. 32 More importantly, GSH, the most important antioxidant in the mammalian liver, is synthesized exclusively in the hepatocytes. 33 GSH plays an important role in direct scavenging of reactive oxygen species (ROS), nitric oxide (NO), and reactive nitrogen species (RNS) with concomitant protection of the electron transport chain, DNA, lipids, and proteins. 34 Observations in our study on the antioxidant effects of glycine corroborate an earlier report of Senthilkumar et al, 35 who reported that the administration of glycine attenuated alcohol‐induced toxicity by inhibiting oxidative stress and elevating enzymatic and non‐enzymatic antioxidants such as GSH, GPx, SOD, and CAT in the liver. Moreover, our findings revealed that liver function tests including AST, ALT, and ALP assays were significantly lowered in the glycine‐treated rats with the corresponding attenuation of pathologic lesions. The serum levels of AST, ALT, and ALP are useful biomarkers of liver injury. 36 Furthermore, various authors have indicated the role of glycine as a potent hepato‐protectant. 37 , 38 , 39 In experimental liver transplantation, glycine was shown to prevent the activation of Kupffer cells and reduce ischemia/reperfusion injury (IRI) in the liver, thus proving to be safe and hepatoprotective. 40

In the present study, glycine demonstrated a potent nephroprotective effect against CoCl2‐induced nephrotoxicity manifested as patchy tubular epithelial necrosis, tubular epithelial degeneration, and periglomerular inflammation in rats exposed to CoCl2 without glycine treatment. Interestingly, the observed lesions were absent in rats treated with glycine. However, the absence of observable pathologic lesions in the kidney of glycine‐treated rats could be due to the attenuation of oxidative stress as indicated by a reduction in MDA and H2O2 contents, as well as heightened activities of GSH, GPx, GST, and SOD. The nephroprotective effects of glycine have also been reported by various authors. For instance, Shafiekhani et al 41 reported a mitigation of lead‐induced renal injury in glycine‐treated rats, with the alleviation of oxidative stress suggested as the most probable mechanism of action, thus corroborating our observations on the preservation of total antioxidant capacity in rats treated with glycine following CoCl2 exposure.

The proposition that glycine is a potent renoprotective agent was further strengthened by our results on the immunohistochemical localization of renal NGAL and renal podocin, which are well characterized novel biomarkers of renal pathologies. 42 , 43 NGAL, a small protein (25 kDa) produced by epithelial cells and neutrophils in most tissues, is a novel biomarker of tubular injury of the kidney and has been suggested to be a valuable diagnostic tool in clinical management of acute and chronic renal diseases. 44 , 45 In the present study, immunohistochemical localization of NGAL in the CoCl2‐exposed rats without glycine treatment was higher than in rats treated with glycine. Thus, our findings suggest that glycine offered prevention of renal damage particularly on the renal epithelial cells. This observation corroborates an earlier report of Wang et al 8 that the protective effect of glycine on renal oxidative stress and structural damage was strongly associated with the potentiation of GSH synthesis with concomitant attenuation of NGAL expression in rats. The podocyte protein known as podocin is essential for the maintenance of structural integrity of slit diaphragm in renal tissues. However, altered expressions of podocin has been associated with several proteinuric glomerular diseases. 46 Interestingly, the results of urinalysis showed that rats exposed to CoCl2 intoxication without glycine treatment tested positive for the presence of protein and ketone bodies, unlike the rats treated with glycine. In an earlier report, podocytes exposed to CoCl2 lost their arborized morphology and cell–cell connections, and displayed cytoskeletal derangements. 47 Renal damage by CoCl2 toxicity was further corroborated by an exaggerated increase in serum BUN and creatinine. A recent study revealed a positive correlation between renal damage and heightened levels of BUN and creatinine. 48 Therefore, the observed decrease in the expression of podocin in renal tissues of rats treated with glycine in this study suggests a preservation of the structural integrity of renal tissues and therefore indicates that glycine is a potent renoprotective agent against renal toxicities associated with CoCl2 exposures. Recent experimental studies also confirmed high expression of podocin as a novel biomarker of renal damage during acute kidney injury. 49 , 50 , 51

In conclusion, the results of this study clearly demonstrate the protective effects of glycine against tissue injuries and derangement of normal physiological functioning of the hepatic and renal systems in rats. The protective effects are mediated via augmentation of the total antioxidant capacity and upregulation of NGAL and podocin expression in rats. These findings suggest that glycine, the simplest amino acid with antioxidant and anti‐inflammatory activities, should be investigated as a novel hepato‐ and nephron‐protective agent.

AUTHOR CONTRIBUTIONS

All authors contributed to the study conception and design. The authors, Oluwafikemi Temitayo Iji, Oluwaseun Olanrewaju Esan, Temitayo Olabisi Ajibade, Omolola Victoria Awoyomi, Moses Olusola Adetona, Ademola Adetokunbo Oyagabemi, Temidayo Olutayo Omobowale, Momoh Audu Yakubu, and Oluwafemi Omoniyi Oguntibeju designed the experiment. Oluwafikemi Temitayo Iji, Oluwaseun Olanrewaju Esan, Temitayo Olabisi Ajibade, Omolola Victoria Awoyomi, and Moses Olusola Adetona performed the immunohistochemistry and biochemical assays. Oluwafikemi Temitayo Iji, Temitayo Olabisi Ajibade, Omolola Victoria Awoyomi, Ademola Adetokunbo Oyagabemi, Temidayo Olutayo Omobowale, Momoh Audu Yakubu, Evaristus Nwulia, and Oluwafemi Omoniyi Oguntibeju proof‐read and approved the submission.

FUNDING INFORMATION

None.

CONFLICT OF INTEREST STATEMENT

Authors declare no conflict of interest.

Supporting information

Supinfo S1

Click here for additional data file. (19.2KB, docx)

ACKNOWLEDGMENTS

We acknowledge Prof. O.O. Oguntibeju of epartment of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville 7535, South Africa for provision of antibodies.

Iji OT, Ajibade TO, Esan OO, et al. Ameliorative effects of glycine on cobalt chloride‐induced hepato‐renal toxicity in rats. Anim Models Exp Med. 2023;6:168‐177. doi: 10.1002/ame2.12315

DATA AVAILABILITY STATEMENT

Data will be supplied based on request.

REFERENCES

  • 1. Tsun ZY, Possemato R. Amino acid management in cancer. Semin Cell Dev Biol. 2015;43:22‐32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Lukey MJ, Katt WP, Cerione RA. Targeting amino acid metabolism for cancer therapy. Drug Discov. 2017;22:796‐804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Lee DY, Kim EH. Therapeutic effects of amino acids in liver diseases: current studies and future perspectives. J Cancer Prev. 2019;24(2):72‐78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Rom O, Villacorta L, Zhang J, Chen YE, Aviram M. Emerging therapeutic potential of glycine in cardiometabolic diseases: dual benefits in lipid and glucose metabolism. Curr Opin Lipidol. 2018;29(5):428‐432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Ruiz‐Ramírez A, Ortiz‐Balderas E, Cardozo‐Saldan AG, Diaz‐Diaz E, El‐Hafidi M. Glycine restores glutathione and protects against oxidative stress in vascular tissue from sucrose‐fed rats. Clin Sci. 2014;126:19‐29. [DOI] [PubMed] [Google Scholar]
  • 6. Rivera CA, Bradford BU, Hunt KJ, et al. Attenuation of CCl(4)‐induced hepatic fibrosis by GdCl(3) treatment or dietary glycine. Am J Physiol Gastrointest Liver Physiol. 2001;281:G200–7. [DOI] [PubMed] [Google Scholar]
  • 7. Kumar P, Liu C, Hsu JW, et al. Glycine and N‐acetylcysteine (GlyNAC) supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: results of a pilot clinical trial. Clin Transl Med. 2021;11(3):e372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Wang Z, Junqing Z, Limei W, et al. Glycine mitigates renal oxidative stress by suppressing Nox4 expression in rats with streptozotocin‐induced diabetes. J Pharmacol Sci. 2018;137(4):387‐394. [DOI] [PubMed] [Google Scholar]
  • 9. Abdel‐Daim MM, Khalil SR, Awad A, Abu Zeid EH, El‐Aziz RA, El‐Serehy HA. Ethanolic extract of Moringa oleifera leaves influences NF‐κB signaling pathway to restore kidney tissue from cobalt‐mediated oxidative injury and inflammation in rats. Nutrients. 2020;12(4):1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Adolfo FR, do Nascimento PC, Bohrer D, et al. Simultaneous determination of cobalt and nickel in vitamin B12 samples using high resolution continuum source atomic absorption spectrometry. Talanta. 2016;147:241‐245. [DOI] [PubMed] [Google Scholar]
  • 11. O'Leary F, Samman S. Vitamin B12 in health and disease. Nutrients. 2010;2(3):299‐316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Mahey S, Kumar R, Sharma M, Kumar V, Bhardwaj R. A critical review on toxicity of cobalt and its bioremediation strategies. SN Appl Sci. 2020;2:1279. [Google Scholar]
  • 13. Permenter MG, Dennis WE, Sutto TE, Jackson DA, Lewis JA, Stallings JD. Exposure to cobalt causes transcriptomic and proteomic changes in two rat liver derived cell lines. PloS One. 2013;8(12):e83751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Ajibade AJ, Ogunmola IA, Okeleye AE. Ameliorative effects of Moringa oleifera leaf extract on the cobalt chloride‐induced liver damage in adult wistar rats. Sys Rev Pharm. 2021;12(12):3865‐3871. [Google Scholar]
  • 15. Venkatraman V, Wong MK, Shalita C, Parente B, Lad SP. Cobalt‐induced toxicity and spasticity secondary to hip arthroplasty: case report and review of the literature. Cureus. 2020;12(12):e12368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Liu YK, Xu H, Liu F, Tao R, Yin J. Effects of serum cobalt ion concentration on the liver, kidney and heart in mice. Orthop Surg. 2010;2(2):134‐140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Mathur N, Jain GC, Geeta P. Hepatotoxicity of cobalt chloride in albino rat. Pharmacol. 2011;2:179‐185. [Google Scholar]
  • 18. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med. 1963;61:882‐888. [PubMed] [Google Scholar]
  • 19. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical role as a component of glutathione peroxidase. Science. 1973;179(4073):588‐590. [DOI] [PubMed] [Google Scholar]
  • 20. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247(10):3170‐3175. [PubMed] [Google Scholar]
  • 21. Habig WH, Pabst MJ, Jakoby WB. Glutathione S‐transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130‐7139. [PubMed] [Google Scholar]
  • 22. Wolff SP. Ferrous ion oxidation in the presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Meth Enzymol. 1994;233:182‐189. [Google Scholar]
  • 23. Varshney R, Kale RK. Effects of calmodulin antagonists on radiation‐induced lipid peroxidation in microsomes. Int J Radiat Biol. 1990;58(5):733‐743. [DOI] [PubMed] [Google Scholar]
  • 24. Drury RA, Wallington EA, Cancerson R. Carlton's Histopathological Techniques. 4th ed. Oxford University Press; 1976. [Google Scholar]
  • 25. Oyagbemi AA, Omobowale TO, Awoyomi OV, et al. Cobalt chloride toxicity elicited hypertension and cardiac complication via induction of oxidative stress and upregulation of COX‐2/Bax signaling pathway. Hum Exper Toxicol. 2019;38(9):519‐532. [DOI] [PubMed] [Google Scholar]
  • 26. Ali AAM, Mohamed HRH. Genotoxicity and oxidative stress induced by the orally administered nanosized nickel and cobalt oxides in male albino rats. J Basic Applied Zool. 2019;80:2. [Google Scholar]
  • 27. Salloum Z, Lehoux EA, Harper ME, Catelas I. Effects of cobalt and chromium ions on oxidative stress and energy metabolism in macrophages in vitro. J Orthop Res. 2018;36(12):3178‐3187. [DOI] [PubMed] [Google Scholar]
  • 28. Kubrak OI, Husak VV, Rovenko BM, Storey JM, Storey KB, Lushchak VI. Cobalt‐induced oxidative stress in brain, liver and kidney of goldfish Carassius auratus . Chemosphere. 2011;85(6):983‐989. [DOI] [PubMed] [Google Scholar]
  • 29. Cichoż‐Lach H, Michalak A. Oxidative stress as a crucial factor in liver diseases. World J Gastroenterol. 2014;20(25):8082‐8091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Kireev R, Bitoun S, Cuesta S, et al. Melatonin treatment protects liver of Zucker rats after ischemia/reperfusion by diminishing oxidative stress and apoptosis. Eur J Pharmacol. 2013;701:185‐193. [DOI] [PubMed] [Google Scholar]
  • 31. Sumida Y, Naito Y, Tanaka S, et al. Long‐term (>=2 yr) efficacy of vitamin E for non‐alcoholic steatohepatitis. Hepatogastroenterol. 2013;60:1445‐1450. [DOI] [PubMed] [Google Scholar]
  • 32. Chen CY, Wang BT, Wu ZC, et al. Glycine ameliorates liver injury and vitamin D deficiency induced by bile duct ligation. Clin Chim Acta. 2013;420:150‐154. [DOI] [PubMed] [Google Scholar]
  • 33. Kalinina E, Novichkova M. Glutathione in protein redox modulation through S‐glutathionylation and S‐nitrosylation. Molecules. 2021;26(2):435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Vairetti M, Di Pasqua LG, Cagna M, Richelmi P, Ferrigno A, Berardo C. Changes in glutathione content in liver diseases: an update. Antioxidants. 2021;10(3):364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Senthilkumar R, Viswanathan P, Nalini N. Effect of glycine on oxidative stress in rats with alcohol induced liver injury. Pharmazie. 2004;59:55‐60. [PubMed] [Google Scholar]
  • 36. Hasan KM, Nasrin Tamanna M, Haque A. Biochemical and histopathological profiling of Wistar rat treated with Brassica napus as a supplementary feed. Food Sci Hum Wellness. 2018;7(1):77‐82. [Google Scholar]
  • 37. Heidari R, Vahid G, Hamidreza M, et al. Mitochondria protection as a mechanism underlying the hepatoprotective effects of glycine in cholestatic mice. Biomed Pharmacother. 2018;2018:1086‐1095. [DOI] [PubMed] [Google Scholar]
  • 38. Parra‐Vizuet J, Camacho‐Luis A, Madrigal‐Santillan E, et al. Hepatoprotective effects of glycine and vitamin E, during the early phase of liver regeneration in the rat. Afr J Pharm Pharmacol. 2009;3:384‐390. [Google Scholar]
  • 39. Heidari R, Jamshidzadeh A, Niknahad H, et al. The hepatoprotection provided by taurine and glycine against antineoplastic drugs induced liver injury in an ex vivo model of normothermic recirculating isolated perfused rat liver. Trends in Pharmaceut Sci. 2016;2(1):59‐76. [Google Scholar]
  • 40. Nickkholgh A, Schemmer P, Polychronidis G, et al. Glycine is graft protective and improves kidney function after liver transplantation: data from HEGPOL‐trial. Am J Transplant. 2016;16(suppl 3):Abstract 500. [Google Scholar]
  • 41. Shafiekhani M, Ommati MM, Azarpira N, Heidari R, Salarian AA. Glycine supplementation mitigates lead‐induced renal injury in mice. J Exp Pharmacol. 2019;11:15‐22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Singer E, Markó L, Paragas N, et al. Neutrophil gelatinase‐associated lipocalin: pathophysiology and clinical applications. Acta Physiol. 2013;207(4):663‐672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Pereira LHM, da Silva CA, Monteiro MLGR, Araújo LS, Rocha LP. Reis MBdR podocin and uPAR are good biomarkers in cases of Focal and segmental glomerulosclerosis in pediatric renal biopsies. PloS One. 2019;14(6):e0217569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Cowland JB, Sorensen OE, Sehested M. Neutrophil gelatinase‐associated lipocalin is up‐regulated in human epithelial cells by IL‐1 beta, but not by TNF‐alpha. J Immunol. 2003;171:6630‐6639. [DOI] [PubMed] [Google Scholar]
  • 45. Bolignano D, Donato V, Coppolino G, et al. Neutrophil gelatinase–associated lipocalin (NGAL) as a marker of kidney damage. Am J Kidney Dis. 2008;52:595‐605. [DOI] [PubMed] [Google Scholar]
  • 46. Pan QR, Ren YL, Zhu JJ, et al. Resveratrol increases nephrin and podocin expression and alleviates renal damage in rats fed a high‐fat diet. Nutrition. 2014;6(7):2619‐2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Krishnamurthy N, Mukhi D, Mungamuri SK, Pasupulati AK. Stabilization of hypoxia‐inducible factor 1α by cobalt chloride impairs podocyte morphology and slit‐diaphragm function. J Cell Biochem. 2018;120(5):1‐12. [DOI] [PubMed] [Google Scholar]
  • 48. Tian H, Zheng X, Wang H. Isorhapontigenin ameliorates high glucose‐induced podocyte and vascular endothelial cell injuries via mitigating oxidative stress and autophagy through the AMPK/Nrf2 pathway. Int Urol Nephrol. 2023;55(2):423‐436. [DOI] [PubMed] [Google Scholar]
  • 49. Li Y, Chen S, Tan J, et al. Combination therapy with DHA and BMSCs suppressed podocyte injury and attenuated renal fibrosis by modulating the TGF‐β1/Smad pathway in MN mice. Ren Fail. 2023;45(1):2120821. doi: 10.1080/0886022X.2022.2120821 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Zhao L, Han S, Chai C. Huangkui capsule alleviates doxorubicin‐induced proteinuria via protecting against podocyte damage and inhibiting JAK/STAT signaling. J Ethnopharmacol. 2023;306:116150. doi: 10.1016/j.jep.2023.116150 [DOI] [PubMed] [Google Scholar]
  • 51. Gomes AM, Lopes D, Almeida C, et al. Potential renal damage biomarkers in alport syndrome‐A review of the literature. Int J Mol Sci. 2022;23(13):7276. doi: 10.3390/ijms23137276 [DOI] [PMC free article] [PubMed] [Google Scholar]

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