Mol Cell Biochem DOI 10.1007/s11010-015-2402-6
Protective effects of N-acetylcysteine against hyperoxaluria induced mitochondrial dysfunction in male wistar rats Minu Sharma1 • Tanzeer Kaur2 • S. K. Singla1
Received: 29 December 2014 / Accepted: 27 March 2015 Ó Springer Science+Business Media New York 2015
Abstract The purpose of the present study was to evaluate the nephro-protective potential of N-acetylcysteine against hyperoxaluria-induced renal mitochondrial dysfunction in rats. Nine days dosing of 0.4 % ethylene glycol ?1 % ammonium chloride, developed hyperoxaluria in male wistar rats which resulted in renal injury and dysfunction as supported by increased level of urinary lactate dehydrogenase, calcium, and decreased creatinine clearance. Mitochondrial oxidative strain in hyperoxaluric animals was evident by decreased levels of superoxide dismutase, glutathione peroxidase, glutathione reductase, reduced glutathione, and an increased lipid peroxidation. Declined activities of respiratory chain enzymes and tricarboxylic acid cycle enzymes showed mitochondrial dysfunction in hyperoxaluric animals. N-acetylcysteine (50 mg/kg, i.p.), by virtue of its –SH reviving power, was able to increase the glutathione levels and thus decrease the oxidative stress in renal mitochondria. Hence, mitochondrial damage is, evidently, an essential event in ethylene glycol-induced hyperoxaluria and N-acetylcysteine presented itself as a safe and effective remedy in combating nephrolithiasis.
Abbreviations COM Calcium oxalate monohydrate DTNB 5,5-Dithiobis-2-nitrobenzoic acid EG Ethylene glycol ETC Electron transport chain GR Glutathione reductase GSH Reduced glutathione GPx Glutathione peroxidase i.p. Intraperitoneal LDH Lactate dehydrogenase LPO Lipid peroxidation NP-SH Non-protein thiols MDA Malondialdehyde Mn-SOD Manganese superoxide dismutase NADH Nicotinamide adenine dinucleotide NAC N-acetylcysteine ROS Reactive oxygen species T-SH Total thiols
Introduction Keywords Mitochondria Hyperoxaluria Oxidative stress N-acetylcysteine Vitamin E Antioxidant
& S. K. Singla [email protected] 1
Department of Biochemistry, Panjab University, Chandigarh 160014, India
2
Department of Biophysics, Panjab University, Chandigarh, India
Nephrolithiasis is crystal deposition in the kidney resulting from an alteration of the normal crystallization conditions of urine in the urinary tract [1]. The risk of developing nephrolithiasis in adults shown to be higher in the western hemisphere (5–9 % in Europe, 12 % in Canada, and 13–15 % in the USA) than in the eastern hemisphere (1–5 %), although the highest risks have been reported in some Asian countries such as Saudi Arabia (20.1 %) with lifetime recurrence rates of up to 50 % [2]. The pathogenesis of calcium oxalate stone formation is a multistep process and essentially includes nucleation, crystal growth,
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crystal aggregation, and crystal retention. It is a multifactorial disease owing to multiple genetic or environmental factors that regulates calcium salt precipitation in the urinary system. Despite detection of urinary stones hundreds of years ago, their pathogenesis & prevention/cure are not fully understood [3]. Hyperoxaluria is a major risk factor of human idiopathic calcium oxalate disease and leads to increased calcium oxalate supersaturation and calcium oxalate stone formation [4]. In renal epithelial cell cultures, oxalate toxicity is accompanied by the generation of reactive oxygen species (ROS) [5–8]. High oxalate concentration imposes oxidative stress on renal cells by stimulating the accretion of lipid peroxides while decreasing the accessibility of major cellular antioxidants, such as reduced glutathione [9]. Inflammation and intracellular morphological changes via oxidative stress are substantive to renal calcium crystallization [10]. The generation of ROS in renal tubular cells exposed to COM crystals has been reported to be induced via mitochondrial collapse [11]. Mitochondria, being the site of aerobic metabolism, are a major source of intracellular ROS under conditions of increased stress [12]. Thus, in addition to oxidative stress and renal tubular cell injury, mitochondria may be the key mediator in oxalateinduced renal cell death [8, 13]. Maintenance of sufficient thiol–disulfide redox status in mitochondria is vital to protect against the damaging effects of ROS [9]. Thus, therapies based on augmenting mitochondrial GSH concentration might be highly beneficial in conditions involving increased mitochondrial ROS production. N-acetylcysteine (NAC), a glutathione precursor, acts as a potent antioxidant by restoring the pool of intracellular reduced glutathione [14]. The benefit of N-acetylcysteine in the prevention of hyperoxaluria-induced nephrolithiasis was earlier reported by Bijarnia et al. [15]. NAC was shown to ameliorate the deleterious effects of inferior vena cava occlusion, due to its scavenging of free radicals and Nitric oxide potentiating capabilities in rats [16]. Studies have shown N-acetylcysteine to exert a potent protective effect on nephrotoxicity associated with gentamicin treatment [17]. NAC was observed to improve kidney function, and reduce renal interstitial inflammation, in rats subjected to renal ischemia reperfusion [18]. Recently, E. Park et al. demonstrated that the protective effect of NAC was due to suppression of mitochondrial dysfunction-mediated apoptosis in Monosodium glutamate-induced cytotoxicity [19]. Hence, the present study was aimed to test the hypothesis if hyperoxaluria-induced oxidative stress results from mitochondrial oxidative stress and dysfunctions leading to renal injury and to evaluate the potential beneficial effect of NAC in preventing hyperoxaluria-induced alterations.
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Materials and methods Chemicals All the chemicals used in the study were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA), Merck (Mumbai, India), and Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). NAC was acquired from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Kits for calcium, creatinine, and LDH estimation were obtained from ERBA Diagnostics Mannheim GmbH (Mannheim/ Germany).
Animal model Male Wistar rats weighing between 120 and 150 g (2–3 month of age) were procured from the Central Animal House of Punjab University. The animals were maintained under standard conditions of humidity, temperature (25 ± 2 °C), and light (12 h light/12 h dark). They were fed standard rat pelleted diet (Ashirwad Industries, India) and were allowed free access to water. The animals were acclimatized to the local vivarium for a week prior to the experimental study. The procedures followed were approved by the Institutional Animal Ethics Committee and were in accordance with the Guidelines for Humane Use and Care of Laboratory Animals (IAEC/222/dated 10/02/ 2012).
Experimental design The animals were randomly segregated into the following four groups with each group having 6–8 animals. Group I (CONTROL) Rats were given intraperitoneal injection of normal saline. Group II (HYPEROXALURIC) Rats received 0.4 % ethylene glycol and 1 % ammonium chloride in drinking water for 9 days to induce hyperoxaluria. Group III (NAC TREATED) Rats received 0.4 % ethylene glycol and 1 % ammonium chloride in drinking water along with N-acetylcysteine (NAC) (50 mg/kg body weight, i.p. per day) for 9 days. Group IV (POSITIVE CONTROL) Rats received 0.4 % ethylene glycol and 1 % ammonium chloride in drinking water along with Vitamin E (200 mg/kg body weight, i.p. per day) for 9 days. Food and water consumption was regularly monitored. Rats were weighed daily to check their growth. Six rats from each group were sacrificed on Day 10.
Mol Cell Biochem
Urine collection and analysis
Mitochondrial antioxidant status
On 9th day of experiment, the rats were placed in metabolic cages and urine was collected for 24 h period in 20 ll of 20 % sodium azide as a preservative. After determining volume and the pH, aliquots of urine for various assays were taken. Urine was examined by light microscopy to analyze crystalluria.
The assay for Superoxide dismutase (Mn-SOD) was performed according to the method of Kono [25]. Glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler [26]. Glutathione reductase activity was assayed by the method of Carlberg and Mannervik [27]. Enzyme activity was calculated using the molar extinction coefficient of NADPH (6.22 9 106 M/cm). The results were expressed as nano moles of NADPH oxidized/min/mg protein.
Biochemical assays in urine and serum Urinary oxalate level was quantified by the colorimetric method as described by Hodgkinson and Williams [20]. Urinary lactate dehydrogenase was measured by a decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit of LDH caused the oxidation of one micromole of NADH per minute at 25 °C and pH 7.3, under the specified conditions [21]. Calcium levels in urine were estimated by calcium estimation kit (OCPC method). Creatinine, in both serum and urine, was estimated by the creatinine estimation kit (Jaffe’s method). Creatinine clearance was calculated according to standard clearance formula C = U/S 9 V, where U is the urinary concentration of creatinine, S is the concentration of creatinine in the serum, and V is the urinary volume in ml/min. Mitochondrial parameters Isolation of mitochondria The kidney was washed in saline at 4 °C, trimmed of adipose and connective tissues, weighed, and homogenized in a Potter/Elvehjem homogenizer, (10 % w/v) in buffer containing 0.25 M sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1 % bovine serum albumin pH 7.2. The whole homogenate was centrifuged at 10009g for 5 min to remove the nuclear fraction and cell debris. Mitochondrial pellet was obtained by centrifuging the post-nuclear supernatant at 14,0009g for 20 min. Mitochondrial pellet was washed thrice with 1.15 % potassium chloride solution and finally suspended in 0.25 M sucrose solution. The purity of mitochondrial preparation was checked by measuring the activity of cytochrome oxidase (complex IV) [22] and citrate synthase [23]. All operations were performed at 4 °C. The mitochondrial fraction was used for the following biochemical studies.
Mitochondrial reactive oxygen species Reactive oxygen species production in isolated mitochondria was measured using 20 ,70 -dichloro-dihydrofluoresceindiacetate (DCF-DA), a non fluorescent dye cleaved to fluorescent by-product by reactive oxygen species, particularly hydrogen peroxide. Study was carried out using a method as described by Wang and Joseph [28]. The results were expressed as p mol/min/mg protein. Mitochondrial thiols Total thiols (T-SH) were determined in the mitochondria according to the method of Sedlak and Hanus [29]. The results were expressed as nmol T-SH/mg protein. Nonprotein thiols (NP-SH) or GSH were quantified as previously described by Roberts and Francetic [30]. Results were expressed as nmol GSH/mg protein. Levels of protein thiols (P-SHs) were calculated from the difference between the values of total thiols and NP-SH levels. Respiratory chain complexes Nicotinamide adenine dinucleotide (NADH) dehydrogenase activity was measured spectrophotometrically as described by King and Howard [31]. Results were expressed as nmol cytochrome c reduced/min/mg protein, using molar extinction coefficient of reduced cytochrome c at 550 nm (19.6 mM/cm). The activity of succinate dehydrogenase was assayed by following the method described by King et al. [32]. The results were expressed as nmol succinate oxidized/min/mg protein. Mitochondrial cytochrome oxidase was assayed according to Sottocasa et al. [22]. Cytochrome oxidase activity was expressed as lmol cytochrome c oxidized/min/mg protein, using molar extinction coefficient of cytochrome c (19.6 mM/cm).
Mitochondrial lipid peroxidation assay MTT assay Mitochondrial lipid peroxidation was determined by the procedure of Buege and Aust [24]. The results were expressed in nmoles of MDA mg -1 protein.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) reduction was used to assess the
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activity of the mitochondrial dehydrogenases in isolated mitochondria as described by Liu et al. [33]. The results were expressed as lg of formazan formed min-1 mg -1 protein. TCA cycle enzymes Citrate synthase was assayed by the method of Kirby et al. [23]. The results were expressed as nmol of DTNB reduced/min/mg protein. Aconitase enzymatic activity was assayed by the method of Fansler and Lowenstein [34]. The results were expressed as nmol of cis Aconitase disappear/ min/mg protein. The activity of isocitrate dehydrogenase (ICDH) was determined by the method of Bergmeyer and Bernt [35].The results were expressed as nmol NADPH formed/min/mg protein. The activity of malate dehydrogenase (MDH) was assayed by the method of Robinson et al. [36]. The results were expressed as n mol NADH oxidized/min/mg protein.
IV, but there were no significant fluctuations among the three groups (p [ 0.05) (Table 1). A significant increase in urinary excretion of calcium was noted in hyperoxaluria group, whereas rats treated with NAC (group III) and Vitamin E (group IV) showed significant decrease in urinary excretion of calcium (Table 1). As shown in Table 1, animals of group II (hyperoxaluric) showed a decline of creatinine clearance by 61.3 % as compared to control animals. NAC-treated animals (group III) showed an increase of 129.6 % in creatinine clearance, whereas vitamin E treatment (group IV) showed only 50.4 % increase in creatinine clearance as compared to hyperoxaluric group. Figure 1 shows significant increase (313.14 %) in urinary Lactate dehydrogenase (LDH) activity in the group II rats as compared to the untreated controls. NAC treatment (group III) resulted in 49.49 % decrease in the urinary LDH activity as compared to hyperoxaluric animals. Vitamin E treatment (group IV) showed 33.19 % decrease in LDH activity as compared to hyperoxaluric group.
Urinary crystal study A drop of urine was spread on a glass slide and visualized under polarized light using Leica DM3000 light microscope. Histopathological studies The kidneys were removed and its transverse sections were fixed in 10 % buffered formalin solution (pH 7). The tissues were dehydrated and embedded with paraffin wax (M.P. 68 °C). The paraffin sections were then cut and finally stained with Delafield’s Hematoxylin and Eosin staining (H & E staining), viewed under polarized light using Leica DM3000 light microscope. Statistical analysis The results are expressed as mean ± standard deviation (SD) for six animals in each group. All analysis for statistical significance was performed using Graph Pad PrismÒ software (Graph Pad Software, San Diego, CA). Data were evaluated by one-way or two-way analysis of variance (ANOVA). p values \0.05 were accepted to be statistically significant.
Mitochondrial oxidative stress and antioxidant status Mitochondrial lipid peroxidation (LPO) was used as an index of mitochondrial oxidative stress. Induction of hyperoxaluria leads to a significant increase (78.81 %) in mitochondrial LPO in renal tissue of group II animals as compared to control animals (Table 2). Vitamin E treatment (group IV) decreased mitochondrial LPO by 16.80 %. The administration of NAC (group III) significantly attenuated (30.02 %) hyperoxaluria-induced LPO in mitochondria. The activities of antioxidant enzymes i.e., superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were decreased significantly (p \ 0.01) in hyperoxaluric (group II) animals as compared to control animals. However, supplementation of NAC in group III and Vitamin E in group IV significantly increased the activities of antioxidant enzymes in renal mitochondria of hyperoxaluric animals (Table 2). The data from Fig. 2, clearly show that mitochondria from hyperoxaluric kidney had a significantly higher rate of ROS production (167.14 %) compared to the mitochondria from control group. NAC supplementation, on the other hand, was found to suppress (50.37 %) the hyperoxaluria-induced mitochondrial ROS production.
Results Mitochondrial thiols Urinary parameters There was a marked increase in urinary excretion of oxalate in rats treated with EG ? NH4Cl in groups II, III, and
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Total thiols level was found to be significantly reduced by 55.50 % in hyperoxaluric (group II) animals as compared to control animals (Fig. 3). NAC supplementation resulted
Mol Cell Biochem Table 1 Effect of N-acetylcysteine (NAC) and Vitamin E administration on urine parameters in EG ? NH4Cl treated (Hyperoxaluric) rats Control I
EG ? NH4Cl II
EG ? NH4Cl ?NAC III
EG ? NH4Cl ? Vitamin E IV
Urine oxalate (mg/24 h)
1.6 ± 0.04
6.5 ± 0.51*
5.8 ± 0.23*
5.9 ± 0.13*
Urine calcium (mg/24 h)
1.72 ± 0.32
2.23 ± 0.08*
1.75 ± 0.21##
1.97 ± 0.11#
Creatinine clearance (ml/min)
3.23 ± 0.4
1.25 ± 0.2*
2.87 ± 0.3##
1.88 ± 0.4#
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
MTT reduction was significantly inhibited by 42.69 % in group II animals as compared to group I. NAC treatment (group III) increased MTT reduction by 56.32 %, whereas Vitamin E treatment (group IV) increased MTT reduction only by 29.64 % as compared to group II animals (Table 3). Mitochondrial tricarboxylic acid cycle (TCA) enzymes
Fig. 1 Effect of N-acetylcysteine (NAC) and Vitamin E administration on lactate dehydrogenase (LDH) levels in urine of EG ? NH4Cl treated (Hyperoxaluric) rats. Values are expressed as mean ± SD; n = 6. *Significantly different from control group (p \ 0.01). #Significantly different from EG ? NH4Cl group (p \ 0.05). ## Significantly different from EG ? NH4Cl group (p \ 0.01)
in increased level of total thiols (70.59 %) in group III animals as compared to hyperoxaluric animals (group II). Vitamin E treatment increased the level of total thiols by only 13.4 % as compared to group II animals. A significant decrease in non-protein thiols (GSH) (57.05 %) was observed in group II animals as compared to group I. NAC supplementation significantly increased the non-protein thiols (GSH) by 95.06 % in hyperoxaluric animals (Fig. 3). Mitochondrial respiratory chain complexes Significant reduction (p \ 0.01) in the activities of respiratory chain complexes was observed in hyperoxaluric animals (group II). Administration of NAC (group III) significantly (p \ 0.01) increased the activities of these enzymes in renal mitochondria of hyperoxaluric animals (Table 3).
Table 4 depicts a significant decrease (p \ 0.01) in the activities of TCA enzymes in group II as compared to group I. NAC effectively abrogated the decrease in enzyme activities (p \ 0.01). Vitamin E also increased the activities of TCA cycle enzymes in group IV in comparison to group II animals. Urinary crystals Polarization light microscopic observations revealed that urine of control animals was devoid of any crystal (Fig. 4a). Figure 4b revealed the presence of aggregated calcium oxalate monohydrate (COM) crystals (dumbbellshaped) in hyperoxaluric rat urine. NAC treatment showed more effective reduction in number of CaOx crystals in hyperoxaluric rat urine as compared to Vitamin E treatment as shown in Fig. 4c, d, respectively. Histopathological analysis Renal histology of control animals (Fig. 5a) showed normal glomeruli structure with lobular organization and a flat epithelial lining the glomerular capsule. Renal tubules encompass distinct lumen and are lined by thick cubic epithelium, whereas animals exposed to EG ? NH4Cl (Fig. 5b) showed shrinkage of glomeruli and increased urinary space. However, histological analysis of NAC administered hyperoxaluric rats (Fig. 5c) showed better lobular organization of glomeruli with no shrinkage and urinary space comparable to control group but renal tubular morphology was not significantly improved.
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Mol Cell Biochem Table 2 Effect of N-acetylcysteine (NAC) and Vitamin E administration on renal mitochondrial Lipid peroxidation (LPO), Superoxide dismutase (SOD), Glutathione peroxidase (GPx) and Glutathione reductase (GR) in EG ? NH4Cl treated (Hyperoxaluric) rats Control I LPO (nmol MDA/mg protein)
2.03 ± 0.15
EG ? NH4Cl ? NAC III
EG ? NH4Cl ? Vitamin E IV
3.63 ± 0.45*
2.54 ± 0.23##
3.02 ± 0.41#
##
9.23 ± 0.52#
52.24 ± 8.41## 16.93 ± 1.06##
46.52 ± 6.02# 13.53 ± 1.16#
EG ? NH4Cl II
SOD (U/mg protein)
15.35 ± 0.56
8.67 ± 0.41*
GPx (nmol of NADPH oxidized/min/mg protein) GR (nmol of NADPH oxidized/min/mg protein)
57.86 ± 10.01 18.34 ± 1.02
41.24 ± 5.23* 11.51 ± 0.95*
12.21 ± 0.34
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
Fig. 2 Effect of N-acetylcysteine (NAC) and Vitamin E administration on mitochondrial ROS production in EG ? NH4Cl treated (Hyperoxaluric) rats. Values are expressed as mean ± SD; n = 6. *Significantly different from control group (p \ 0.01). #Significantly different from EG ? NH4Cl group (p \ 0.05). ##Significantly different from EG ? NH4Cl group (p \ 0.01)
Vitamin E administration (Fig. 5d) moderately improved the renal cell morphology.
Discussion The purpose of the present study was to explore the role of NAC in ameliorating the hyperoxaluria-induced mitochondrial dysfunction. It was observed that there was an increase in oxalate levels after ethylene glycol and ammonium chloride administration. Hyperoxaluria resulted in oxidative imbalance and lipid peroxidation in renal mitochondria. Dwindling activities of respiratory chain enzymes and TCA cycle enzymes showed mitochondrial dysfunction in hyperoxaluric animals. The administration of NAC protected mitochondria from hyperoxaluria-induced oxidative damage by restoring the antioxidant potential as manifested by increased thiol levels.
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Fig. 3 Effect of N-acetylcysteine (NAC) and Vitamin E administration on renal mitochondrial total, Non protein thiols (GSH) and protein thiols in EG ? NH4Cl treated (Hyperoxaluric) rats. Values are expressed as mean ± SD; n = 6. *Significantly different from control group (p \ 0.01), #Significantly different from EG ? NH4Cl group (p \ 0.05). ##Significantly different from EG ? NH4Cl group (p \ 0.01)
Hyperoxaluria-induced generation of oxidative stress is considered as the initial trigger for a vicious cycle of nephrolithiasis [37–39]. Free radical-mediated inflammation by hyperoxaluria has been supported by the beneficial effects of free radical scavengers viz. vitamin E, glutathione, mannitol, and allopurinol [40–42]. In the present study, Vitamin E was used as a positive control as it was shown to have antioxidant potential in combating hyperoxaluria-induced renal damage in a number of studies [42, 43]. Ethylene glycol ingestion has been widely used as an experimental model for the study of nephrolithiasis [44– 46]. Nine days dosing of 0.4 % EG ? 1 % NH4Cl to male wistar rats showed significant increase in the urinary oxalate level, successfully induced hyperoxaluria and produced remarkable renal dysfunction, characterized by significant decrease in the creatinine clearance, which is often used as a rough measurement of glomerular filtration rate. NAC significantly diminished the oxidative damage
Mol Cell Biochem
induced by ethylene glycol on renal tissue of hyperoxaluric animals as indicated by decreased levels of LDH, an indicator of renal tissue damage. [47, 48]. These results corroborate findings of a previous study by our group which have proved that N-acetylcysteine significantly reduced hyperoxaluria-caused oxidative stress by reducing lipid peroxidation, restoring antioxidant enzymes activity in kidney tissue, followed by reduction in impairment of renal functioning. [15] In the present study, NAC supplementation was able to restore thiol levels in hyperoxaluric rats. This may be attributed to the fact that NAC is deacetylated into cysteine, which is rapidly oxidized to cystine and then transported into the cells where it is reduced to cysteine for GSH synthesis, hence accelerating cellular recovery [49]. In a previous study by Muthukumar and Selvam [9], supplementation of (GME) glutathione mono ethyl ester, along with EG was found to dramatically restore the concentration of renal cell total thiols and GSH to normal.
Mitochondria have an impressive array of antioxidant enzymes like superoxide dismutase and glutathione peroxidase that contain critical sulfhydryl groups in their active site for efficient functioning [50]. However, under hyperoxaluric conditions attack by free radicals on these sulfhydryl groups might have decreased their activity. The decrease in glutathione reductase might be due to reduction in the availability of its cofactor, NADPH [51]. NAC treatment, on the other hand, was able to protect these crucial mitochondrial antioxidant enzymes from hyperoxaluria-induced oxidative damage. The restoration of MnSOD activity by NAC might involve induction of enzyme as reducing agents have been shown to induce Mn-SOD expression through activation of NF-jB [52]. Mitochondrial GSH pool is most crucial in maintaining mitochondrial structure and function. Mitochondria solely rely on GSH and GSH-dependent peroxidases and Mn-SOD for the removal of toxic radical intermediates [9]. GSH depletion could lead to an oxidative stress condition which in
Table 3 Effect of N-acetylcysteine (NAC) and Vitamin E administration on activities of renal mitochondrial Respiratory chain enzymes and MTT reduction rate in the EG ? NH4Cl treated (Hyperoxaluric) animals Control I Complex I (nmol Cytochrome c reduced/min/mg protein) Complex II (nmol succinate oxidized/min/mg protein) Complex IV (lmol Cytochrome c oxidized/min/mg protein)
EG ? NH4Cl II
EG ? NH4Cl ? NAC III
EG ? NH4Cl ? Vitamin E IV
34.84 ± 4.76
22.41 ± 3.59*
30.63 ± 2.91##
26.21 ± 1.50#
139.32 ± 16.92
97.21 ± 11.01*
125.17 ± 12.63##
108.45 ± 12.17#
153.05 ± 14.57
113.49 ± 10.82*
141.72 ± 18.01##
120.12 ± 16.01#
8.83 ± 0.61
5.06 ± 0.64*
7.91 ± 0.52##
6.56 ± 0.45#
MTT reduction (lg formazon formed/min/mg protein)
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
Table 4 Effect of N-acetylcysteine (NAC) and Vitamin E administration on the activities of renal mitochondrial TCA cycle enzymes in EG ? NH4Cl treated (Hyperoxaluric) animals Control I
EG ? NH4Cl II
EG ? NH4Cl ? NAC III
Citrate synthase (n mol of DTNB reduced/min/mg protein)
16.51 ± 0.12
10.28 ± 0.39*
15.42 ± 0.63
##
12.24 ± 0.87#
Aconitase (n mol of cis Aconitate disappear/min/mg protein)
28.73 ± 1.16
17.42 ± 0.98*
25.21 ± 1.22
##
19.67 ± 1.64
Isocitrate dehydrogenase (n mol of NADPH formed/min/mg protein)
11.83 ± 1.46
6.79 ± 0.45*
9.95 ± 1.02
##
8.18 ± 1.12#
5.71 ± 0.89
2.07 ± 0.24*
4.90 ± 0.34
##
2.88 ± 0.38
Malate dehydrogenase (n mol of NADH oxidized/min/mg protein)
EG ? NH4Cl ? Vitamin E IV
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
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Mol Cell Biochem Fig. 4 Crystalluria depicted by polarization micrographs of experimental rat’s urine a Urine of control rats (group I) devoid of any crystals. b Urine of hyperoxaluric rats (group II) showing aggregate of COM crystals (Arrows indicate dumbbell-shaped COM crystals). c Urine of NACtreated rats (group III) showing few crystals. d Urine of Vitamin E treated rats (group IV) showing moderate amount of crystals. Original magnification 1009
Fig. 5 Renal histological analysis, stained with haematoxylin and eosin (original magnification 4009). a Control rats (group I). b Hyperoxaluric rats (group II). c NAC treated hyperoxaluric rats (group III). d Vitamin E treated hyperoxaluric rats (group IV). Arrows indicate Shrinkage of glomerulus and increased urinary space in hyperoxaluric rats
this study is supported by the presence of increased levels of MDA concentration in GSH depleted mitochondrial fractions in hyperoxaluric animals (group II). NAC significantly
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increased mitochondrial NP-SH (GSH) which might play a key role in the protection of mitochondrial components against hyperoxaluria-induced oxidative damage.
Mol Cell Biochem
We observed reduced activities of mitochondrial ETC complexes in hyperoxaluria, while NAC supplementation was observed to moderately reverse the loss. The decreased activities of mitochondrial enzymes in the present study are in agreement with the earlier studies in hyperoxaluric rats [13, 45]. Decrease in mitochondrial thiols following chronic hyperoxaluria might contribute to diminished activities of mitochondrial electron transport chain enzymes. It has also been shown that increased LPO or decrease in the antioxidants is associated with a loss of complex IV activity that consequently leads to mitochondria-dependent apoptosis [53]. Since kidney has a high energy demand, any decline in mitochondrial electron chain complex activities might have severe impact on kidney functions. Therefore, electron respiratory chain components appear to be the main mitochondrial targets of chronic hyperoxaluria. The restoration of mitochondrial enzyme activity by NAC might be the result of increase in the intracellular thiols which might offer protection by scavenging ROS or protecting thiols in mitochondrial proteins or both. The decrease in mitochondrial function in the present study was further evident from the diminished activities of the TCA cycle enzymes like citrate synthase, aconitase, ICDH, and MDH in hyperoxaluric animals. Increase in oxalate load might be the possible reason for the dwindling activities of these enzymes, as reactive oxygen species produced by oxalate/calcium oxalate can inactivate the mitochondrial enzymes [45]. NAC administration was able to reduce oxidative stress in hyperoxaluric mitochondria, this might be attributed to the normalization of activities of TCA enzymes. In conclusion, the present study confirms that mitochondrial oxidative stress and dysfunctions might contribute in part in the pathogenesis of hyperoxaluria-induced nephrolithiasis. In addition, the findings of the present study provide evidence that NAC might exert protective effects in hyperoxaluria through mitochondrial protection that may involve attenuation of oxidative stress. Further studies are needed in order to clarify the valid mechanism underlying these effects. Acknowledgments The financial assistance provided by the University Grants Commission, New Delhi is gratefully acknowledged. Conflict of interest
The authors state no conflict of interest.
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Protective effects of N-acetylcysteine against hyperoxaluria induced mitochondrial dysfunction in male wistar rats Minu Sharma1 • Tanzeer Kaur2 • S. K. Singla1
Received: 29 December 2014 / Accepted: 27 March 2015 Ó Springer Science+Business Media New York 2015
Abstract The purpose of the present study was to evaluate the nephro-protective potential of N-acetylcysteine against hyperoxaluria-induced renal mitochondrial dysfunction in rats. Nine days dosing of 0.4 % ethylene glycol ?1 % ammonium chloride, developed hyperoxaluria in male wistar rats which resulted in renal injury and dysfunction as supported by increased level of urinary lactate dehydrogenase, calcium, and decreased creatinine clearance. Mitochondrial oxidative strain in hyperoxaluric animals was evident by decreased levels of superoxide dismutase, glutathione peroxidase, glutathione reductase, reduced glutathione, and an increased lipid peroxidation. Declined activities of respiratory chain enzymes and tricarboxylic acid cycle enzymes showed mitochondrial dysfunction in hyperoxaluric animals. N-acetylcysteine (50 mg/kg, i.p.), by virtue of its –SH reviving power, was able to increase the glutathione levels and thus decrease the oxidative stress in renal mitochondria. Hence, mitochondrial damage is, evidently, an essential event in ethylene glycol-induced hyperoxaluria and N-acetylcysteine presented itself as a safe and effective remedy in combating nephrolithiasis.
Abbreviations COM Calcium oxalate monohydrate DTNB 5,5-Dithiobis-2-nitrobenzoic acid EG Ethylene glycol ETC Electron transport chain GR Glutathione reductase GSH Reduced glutathione GPx Glutathione peroxidase i.p. Intraperitoneal LDH Lactate dehydrogenase LPO Lipid peroxidation NP-SH Non-protein thiols MDA Malondialdehyde Mn-SOD Manganese superoxide dismutase NADH Nicotinamide adenine dinucleotide NAC N-acetylcysteine ROS Reactive oxygen species T-SH Total thiols
Introduction Keywords Mitochondria Hyperoxaluria Oxidative stress N-acetylcysteine Vitamin E Antioxidant
& S. K. Singla [email protected] 1
Department of Biochemistry, Panjab University, Chandigarh 160014, India
2
Department of Biophysics, Panjab University, Chandigarh, India
Nephrolithiasis is crystal deposition in the kidney resulting from an alteration of the normal crystallization conditions of urine in the urinary tract [1]. The risk of developing nephrolithiasis in adults shown to be higher in the western hemisphere (5–9 % in Europe, 12 % in Canada, and 13–15 % in the USA) than in the eastern hemisphere (1–5 %), although the highest risks have been reported in some Asian countries such as Saudi Arabia (20.1 %) with lifetime recurrence rates of up to 50 % [2]. The pathogenesis of calcium oxalate stone formation is a multistep process and essentially includes nucleation, crystal growth,
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crystal aggregation, and crystal retention. It is a multifactorial disease owing to multiple genetic or environmental factors that regulates calcium salt precipitation in the urinary system. Despite detection of urinary stones hundreds of years ago, their pathogenesis & prevention/cure are not fully understood [3]. Hyperoxaluria is a major risk factor of human idiopathic calcium oxalate disease and leads to increased calcium oxalate supersaturation and calcium oxalate stone formation [4]. In renal epithelial cell cultures, oxalate toxicity is accompanied by the generation of reactive oxygen species (ROS) [5–8]. High oxalate concentration imposes oxidative stress on renal cells by stimulating the accretion of lipid peroxides while decreasing the accessibility of major cellular antioxidants, such as reduced glutathione [9]. Inflammation and intracellular morphological changes via oxidative stress are substantive to renal calcium crystallization [10]. The generation of ROS in renal tubular cells exposed to COM crystals has been reported to be induced via mitochondrial collapse [11]. Mitochondria, being the site of aerobic metabolism, are a major source of intracellular ROS under conditions of increased stress [12]. Thus, in addition to oxidative stress and renal tubular cell injury, mitochondria may be the key mediator in oxalateinduced renal cell death [8, 13]. Maintenance of sufficient thiol–disulfide redox status in mitochondria is vital to protect against the damaging effects of ROS [9]. Thus, therapies based on augmenting mitochondrial GSH concentration might be highly beneficial in conditions involving increased mitochondrial ROS production. N-acetylcysteine (NAC), a glutathione precursor, acts as a potent antioxidant by restoring the pool of intracellular reduced glutathione [14]. The benefit of N-acetylcysteine in the prevention of hyperoxaluria-induced nephrolithiasis was earlier reported by Bijarnia et al. [15]. NAC was shown to ameliorate the deleterious effects of inferior vena cava occlusion, due to its scavenging of free radicals and Nitric oxide potentiating capabilities in rats [16]. Studies have shown N-acetylcysteine to exert a potent protective effect on nephrotoxicity associated with gentamicin treatment [17]. NAC was observed to improve kidney function, and reduce renal interstitial inflammation, in rats subjected to renal ischemia reperfusion [18]. Recently, E. Park et al. demonstrated that the protective effect of NAC was due to suppression of mitochondrial dysfunction-mediated apoptosis in Monosodium glutamate-induced cytotoxicity [19]. Hence, the present study was aimed to test the hypothesis if hyperoxaluria-induced oxidative stress results from mitochondrial oxidative stress and dysfunctions leading to renal injury and to evaluate the potential beneficial effect of NAC in preventing hyperoxaluria-induced alterations.
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Materials and methods Chemicals All the chemicals used in the study were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA), Merck (Mumbai, India), and Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). NAC was acquired from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Kits for calcium, creatinine, and LDH estimation were obtained from ERBA Diagnostics Mannheim GmbH (Mannheim/ Germany).
Animal model Male Wistar rats weighing between 120 and 150 g (2–3 month of age) were procured from the Central Animal House of Punjab University. The animals were maintained under standard conditions of humidity, temperature (25 ± 2 °C), and light (12 h light/12 h dark). They were fed standard rat pelleted diet (Ashirwad Industries, India) and were allowed free access to water. The animals were acclimatized to the local vivarium for a week prior to the experimental study. The procedures followed were approved by the Institutional Animal Ethics Committee and were in accordance with the Guidelines for Humane Use and Care of Laboratory Animals (IAEC/222/dated 10/02/ 2012).
Experimental design The animals were randomly segregated into the following four groups with each group having 6–8 animals. Group I (CONTROL) Rats were given intraperitoneal injection of normal saline. Group II (HYPEROXALURIC) Rats received 0.4 % ethylene glycol and 1 % ammonium chloride in drinking water for 9 days to induce hyperoxaluria. Group III (NAC TREATED) Rats received 0.4 % ethylene glycol and 1 % ammonium chloride in drinking water along with N-acetylcysteine (NAC) (50 mg/kg body weight, i.p. per day) for 9 days. Group IV (POSITIVE CONTROL) Rats received 0.4 % ethylene glycol and 1 % ammonium chloride in drinking water along with Vitamin E (200 mg/kg body weight, i.p. per day) for 9 days. Food and water consumption was regularly monitored. Rats were weighed daily to check their growth. Six rats from each group were sacrificed on Day 10.
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Urine collection and analysis
Mitochondrial antioxidant status
On 9th day of experiment, the rats were placed in metabolic cages and urine was collected for 24 h period in 20 ll of 20 % sodium azide as a preservative. After determining volume and the pH, aliquots of urine for various assays were taken. Urine was examined by light microscopy to analyze crystalluria.
The assay for Superoxide dismutase (Mn-SOD) was performed according to the method of Kono [25]. Glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler [26]. Glutathione reductase activity was assayed by the method of Carlberg and Mannervik [27]. Enzyme activity was calculated using the molar extinction coefficient of NADPH (6.22 9 106 M/cm). The results were expressed as nano moles of NADPH oxidized/min/mg protein.
Biochemical assays in urine and serum Urinary oxalate level was quantified by the colorimetric method as described by Hodgkinson and Williams [20]. Urinary lactate dehydrogenase was measured by a decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit of LDH caused the oxidation of one micromole of NADH per minute at 25 °C and pH 7.3, under the specified conditions [21]. Calcium levels in urine were estimated by calcium estimation kit (OCPC method). Creatinine, in both serum and urine, was estimated by the creatinine estimation kit (Jaffe’s method). Creatinine clearance was calculated according to standard clearance formula C = U/S 9 V, where U is the urinary concentration of creatinine, S is the concentration of creatinine in the serum, and V is the urinary volume in ml/min. Mitochondrial parameters Isolation of mitochondria The kidney was washed in saline at 4 °C, trimmed of adipose and connective tissues, weighed, and homogenized in a Potter/Elvehjem homogenizer, (10 % w/v) in buffer containing 0.25 M sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1 % bovine serum albumin pH 7.2. The whole homogenate was centrifuged at 10009g for 5 min to remove the nuclear fraction and cell debris. Mitochondrial pellet was obtained by centrifuging the post-nuclear supernatant at 14,0009g for 20 min. Mitochondrial pellet was washed thrice with 1.15 % potassium chloride solution and finally suspended in 0.25 M sucrose solution. The purity of mitochondrial preparation was checked by measuring the activity of cytochrome oxidase (complex IV) [22] and citrate synthase [23]. All operations were performed at 4 °C. The mitochondrial fraction was used for the following biochemical studies.
Mitochondrial reactive oxygen species Reactive oxygen species production in isolated mitochondria was measured using 20 ,70 -dichloro-dihydrofluoresceindiacetate (DCF-DA), a non fluorescent dye cleaved to fluorescent by-product by reactive oxygen species, particularly hydrogen peroxide. Study was carried out using a method as described by Wang and Joseph [28]. The results were expressed as p mol/min/mg protein. Mitochondrial thiols Total thiols (T-SH) were determined in the mitochondria according to the method of Sedlak and Hanus [29]. The results were expressed as nmol T-SH/mg protein. Nonprotein thiols (NP-SH) or GSH were quantified as previously described by Roberts and Francetic [30]. Results were expressed as nmol GSH/mg protein. Levels of protein thiols (P-SHs) were calculated from the difference between the values of total thiols and NP-SH levels. Respiratory chain complexes Nicotinamide adenine dinucleotide (NADH) dehydrogenase activity was measured spectrophotometrically as described by King and Howard [31]. Results were expressed as nmol cytochrome c reduced/min/mg protein, using molar extinction coefficient of reduced cytochrome c at 550 nm (19.6 mM/cm). The activity of succinate dehydrogenase was assayed by following the method described by King et al. [32]. The results were expressed as nmol succinate oxidized/min/mg protein. Mitochondrial cytochrome oxidase was assayed according to Sottocasa et al. [22]. Cytochrome oxidase activity was expressed as lmol cytochrome c oxidized/min/mg protein, using molar extinction coefficient of cytochrome c (19.6 mM/cm).
Mitochondrial lipid peroxidation assay MTT assay Mitochondrial lipid peroxidation was determined by the procedure of Buege and Aust [24]. The results were expressed in nmoles of MDA mg -1 protein.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) reduction was used to assess the
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activity of the mitochondrial dehydrogenases in isolated mitochondria as described by Liu et al. [33]. The results were expressed as lg of formazan formed min-1 mg -1 protein. TCA cycle enzymes Citrate synthase was assayed by the method of Kirby et al. [23]. The results were expressed as nmol of DTNB reduced/min/mg protein. Aconitase enzymatic activity was assayed by the method of Fansler and Lowenstein [34]. The results were expressed as nmol of cis Aconitase disappear/ min/mg protein. The activity of isocitrate dehydrogenase (ICDH) was determined by the method of Bergmeyer and Bernt [35].The results were expressed as nmol NADPH formed/min/mg protein. The activity of malate dehydrogenase (MDH) was assayed by the method of Robinson et al. [36]. The results were expressed as n mol NADH oxidized/min/mg protein.
IV, but there were no significant fluctuations among the three groups (p [ 0.05) (Table 1). A significant increase in urinary excretion of calcium was noted in hyperoxaluria group, whereas rats treated with NAC (group III) and Vitamin E (group IV) showed significant decrease in urinary excretion of calcium (Table 1). As shown in Table 1, animals of group II (hyperoxaluric) showed a decline of creatinine clearance by 61.3 % as compared to control animals. NAC-treated animals (group III) showed an increase of 129.6 % in creatinine clearance, whereas vitamin E treatment (group IV) showed only 50.4 % increase in creatinine clearance as compared to hyperoxaluric group. Figure 1 shows significant increase (313.14 %) in urinary Lactate dehydrogenase (LDH) activity in the group II rats as compared to the untreated controls. NAC treatment (group III) resulted in 49.49 % decrease in the urinary LDH activity as compared to hyperoxaluric animals. Vitamin E treatment (group IV) showed 33.19 % decrease in LDH activity as compared to hyperoxaluric group.
Urinary crystal study A drop of urine was spread on a glass slide and visualized under polarized light using Leica DM3000 light microscope. Histopathological studies The kidneys were removed and its transverse sections were fixed in 10 % buffered formalin solution (pH 7). The tissues were dehydrated and embedded with paraffin wax (M.P. 68 °C). The paraffin sections were then cut and finally stained with Delafield’s Hematoxylin and Eosin staining (H & E staining), viewed under polarized light using Leica DM3000 light microscope. Statistical analysis The results are expressed as mean ± standard deviation (SD) for six animals in each group. All analysis for statistical significance was performed using Graph Pad PrismÒ software (Graph Pad Software, San Diego, CA). Data were evaluated by one-way or two-way analysis of variance (ANOVA). p values \0.05 were accepted to be statistically significant.
Mitochondrial oxidative stress and antioxidant status Mitochondrial lipid peroxidation (LPO) was used as an index of mitochondrial oxidative stress. Induction of hyperoxaluria leads to a significant increase (78.81 %) in mitochondrial LPO in renal tissue of group II animals as compared to control animals (Table 2). Vitamin E treatment (group IV) decreased mitochondrial LPO by 16.80 %. The administration of NAC (group III) significantly attenuated (30.02 %) hyperoxaluria-induced LPO in mitochondria. The activities of antioxidant enzymes i.e., superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were decreased significantly (p \ 0.01) in hyperoxaluric (group II) animals as compared to control animals. However, supplementation of NAC in group III and Vitamin E in group IV significantly increased the activities of antioxidant enzymes in renal mitochondria of hyperoxaluric animals (Table 2). The data from Fig. 2, clearly show that mitochondria from hyperoxaluric kidney had a significantly higher rate of ROS production (167.14 %) compared to the mitochondria from control group. NAC supplementation, on the other hand, was found to suppress (50.37 %) the hyperoxaluria-induced mitochondrial ROS production.
Results Mitochondrial thiols Urinary parameters There was a marked increase in urinary excretion of oxalate in rats treated with EG ? NH4Cl in groups II, III, and
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Total thiols level was found to be significantly reduced by 55.50 % in hyperoxaluric (group II) animals as compared to control animals (Fig. 3). NAC supplementation resulted
Mol Cell Biochem Table 1 Effect of N-acetylcysteine (NAC) and Vitamin E administration on urine parameters in EG ? NH4Cl treated (Hyperoxaluric) rats Control I
EG ? NH4Cl II
EG ? NH4Cl ?NAC III
EG ? NH4Cl ? Vitamin E IV
Urine oxalate (mg/24 h)
1.6 ± 0.04
6.5 ± 0.51*
5.8 ± 0.23*
5.9 ± 0.13*
Urine calcium (mg/24 h)
1.72 ± 0.32
2.23 ± 0.08*
1.75 ± 0.21##
1.97 ± 0.11#
Creatinine clearance (ml/min)
3.23 ± 0.4
1.25 ± 0.2*
2.87 ± 0.3##
1.88 ± 0.4#
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
MTT reduction was significantly inhibited by 42.69 % in group II animals as compared to group I. NAC treatment (group III) increased MTT reduction by 56.32 %, whereas Vitamin E treatment (group IV) increased MTT reduction only by 29.64 % as compared to group II animals (Table 3). Mitochondrial tricarboxylic acid cycle (TCA) enzymes
Fig. 1 Effect of N-acetylcysteine (NAC) and Vitamin E administration on lactate dehydrogenase (LDH) levels in urine of EG ? NH4Cl treated (Hyperoxaluric) rats. Values are expressed as mean ± SD; n = 6. *Significantly different from control group (p \ 0.01). #Significantly different from EG ? NH4Cl group (p \ 0.05). ## Significantly different from EG ? NH4Cl group (p \ 0.01)
in increased level of total thiols (70.59 %) in group III animals as compared to hyperoxaluric animals (group II). Vitamin E treatment increased the level of total thiols by only 13.4 % as compared to group II animals. A significant decrease in non-protein thiols (GSH) (57.05 %) was observed in group II animals as compared to group I. NAC supplementation significantly increased the non-protein thiols (GSH) by 95.06 % in hyperoxaluric animals (Fig. 3). Mitochondrial respiratory chain complexes Significant reduction (p \ 0.01) in the activities of respiratory chain complexes was observed in hyperoxaluric animals (group II). Administration of NAC (group III) significantly (p \ 0.01) increased the activities of these enzymes in renal mitochondria of hyperoxaluric animals (Table 3).
Table 4 depicts a significant decrease (p \ 0.01) in the activities of TCA enzymes in group II as compared to group I. NAC effectively abrogated the decrease in enzyme activities (p \ 0.01). Vitamin E also increased the activities of TCA cycle enzymes in group IV in comparison to group II animals. Urinary crystals Polarization light microscopic observations revealed that urine of control animals was devoid of any crystal (Fig. 4a). Figure 4b revealed the presence of aggregated calcium oxalate monohydrate (COM) crystals (dumbbellshaped) in hyperoxaluric rat urine. NAC treatment showed more effective reduction in number of CaOx crystals in hyperoxaluric rat urine as compared to Vitamin E treatment as shown in Fig. 4c, d, respectively. Histopathological analysis Renal histology of control animals (Fig. 5a) showed normal glomeruli structure with lobular organization and a flat epithelial lining the glomerular capsule. Renal tubules encompass distinct lumen and are lined by thick cubic epithelium, whereas animals exposed to EG ? NH4Cl (Fig. 5b) showed shrinkage of glomeruli and increased urinary space. However, histological analysis of NAC administered hyperoxaluric rats (Fig. 5c) showed better lobular organization of glomeruli with no shrinkage and urinary space comparable to control group but renal tubular morphology was not significantly improved.
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Mol Cell Biochem Table 2 Effect of N-acetylcysteine (NAC) and Vitamin E administration on renal mitochondrial Lipid peroxidation (LPO), Superoxide dismutase (SOD), Glutathione peroxidase (GPx) and Glutathione reductase (GR) in EG ? NH4Cl treated (Hyperoxaluric) rats Control I LPO (nmol MDA/mg protein)
2.03 ± 0.15
EG ? NH4Cl ? NAC III
EG ? NH4Cl ? Vitamin E IV
3.63 ± 0.45*
2.54 ± 0.23##
3.02 ± 0.41#
##
9.23 ± 0.52#
52.24 ± 8.41## 16.93 ± 1.06##
46.52 ± 6.02# 13.53 ± 1.16#
EG ? NH4Cl II
SOD (U/mg protein)
15.35 ± 0.56
8.67 ± 0.41*
GPx (nmol of NADPH oxidized/min/mg protein) GR (nmol of NADPH oxidized/min/mg protein)
57.86 ± 10.01 18.34 ± 1.02
41.24 ± 5.23* 11.51 ± 0.95*
12.21 ± 0.34
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
Fig. 2 Effect of N-acetylcysteine (NAC) and Vitamin E administration on mitochondrial ROS production in EG ? NH4Cl treated (Hyperoxaluric) rats. Values are expressed as mean ± SD; n = 6. *Significantly different from control group (p \ 0.01). #Significantly different from EG ? NH4Cl group (p \ 0.05). ##Significantly different from EG ? NH4Cl group (p \ 0.01)
Vitamin E administration (Fig. 5d) moderately improved the renal cell morphology.
Discussion The purpose of the present study was to explore the role of NAC in ameliorating the hyperoxaluria-induced mitochondrial dysfunction. It was observed that there was an increase in oxalate levels after ethylene glycol and ammonium chloride administration. Hyperoxaluria resulted in oxidative imbalance and lipid peroxidation in renal mitochondria. Dwindling activities of respiratory chain enzymes and TCA cycle enzymes showed mitochondrial dysfunction in hyperoxaluric animals. The administration of NAC protected mitochondria from hyperoxaluria-induced oxidative damage by restoring the antioxidant potential as manifested by increased thiol levels.
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Fig. 3 Effect of N-acetylcysteine (NAC) and Vitamin E administration on renal mitochondrial total, Non protein thiols (GSH) and protein thiols in EG ? NH4Cl treated (Hyperoxaluric) rats. Values are expressed as mean ± SD; n = 6. *Significantly different from control group (p \ 0.01), #Significantly different from EG ? NH4Cl group (p \ 0.05). ##Significantly different from EG ? NH4Cl group (p \ 0.01)
Hyperoxaluria-induced generation of oxidative stress is considered as the initial trigger for a vicious cycle of nephrolithiasis [37–39]. Free radical-mediated inflammation by hyperoxaluria has been supported by the beneficial effects of free radical scavengers viz. vitamin E, glutathione, mannitol, and allopurinol [40–42]. In the present study, Vitamin E was used as a positive control as it was shown to have antioxidant potential in combating hyperoxaluria-induced renal damage in a number of studies [42, 43]. Ethylene glycol ingestion has been widely used as an experimental model for the study of nephrolithiasis [44– 46]. Nine days dosing of 0.4 % EG ? 1 % NH4Cl to male wistar rats showed significant increase in the urinary oxalate level, successfully induced hyperoxaluria and produced remarkable renal dysfunction, characterized by significant decrease in the creatinine clearance, which is often used as a rough measurement of glomerular filtration rate. NAC significantly diminished the oxidative damage
Mol Cell Biochem
induced by ethylene glycol on renal tissue of hyperoxaluric animals as indicated by decreased levels of LDH, an indicator of renal tissue damage. [47, 48]. These results corroborate findings of a previous study by our group which have proved that N-acetylcysteine significantly reduced hyperoxaluria-caused oxidative stress by reducing lipid peroxidation, restoring antioxidant enzymes activity in kidney tissue, followed by reduction in impairment of renal functioning. [15] In the present study, NAC supplementation was able to restore thiol levels in hyperoxaluric rats. This may be attributed to the fact that NAC is deacetylated into cysteine, which is rapidly oxidized to cystine and then transported into the cells where it is reduced to cysteine for GSH synthesis, hence accelerating cellular recovery [49]. In a previous study by Muthukumar and Selvam [9], supplementation of (GME) glutathione mono ethyl ester, along with EG was found to dramatically restore the concentration of renal cell total thiols and GSH to normal.
Mitochondria have an impressive array of antioxidant enzymes like superoxide dismutase and glutathione peroxidase that contain critical sulfhydryl groups in their active site for efficient functioning [50]. However, under hyperoxaluric conditions attack by free radicals on these sulfhydryl groups might have decreased their activity. The decrease in glutathione reductase might be due to reduction in the availability of its cofactor, NADPH [51]. NAC treatment, on the other hand, was able to protect these crucial mitochondrial antioxidant enzymes from hyperoxaluria-induced oxidative damage. The restoration of MnSOD activity by NAC might involve induction of enzyme as reducing agents have been shown to induce Mn-SOD expression through activation of NF-jB [52]. Mitochondrial GSH pool is most crucial in maintaining mitochondrial structure and function. Mitochondria solely rely on GSH and GSH-dependent peroxidases and Mn-SOD for the removal of toxic radical intermediates [9]. GSH depletion could lead to an oxidative stress condition which in
Table 3 Effect of N-acetylcysteine (NAC) and Vitamin E administration on activities of renal mitochondrial Respiratory chain enzymes and MTT reduction rate in the EG ? NH4Cl treated (Hyperoxaluric) animals Control I Complex I (nmol Cytochrome c reduced/min/mg protein) Complex II (nmol succinate oxidized/min/mg protein) Complex IV (lmol Cytochrome c oxidized/min/mg protein)
EG ? NH4Cl II
EG ? NH4Cl ? NAC III
EG ? NH4Cl ? Vitamin E IV
34.84 ± 4.76
22.41 ± 3.59*
30.63 ± 2.91##
26.21 ± 1.50#
139.32 ± 16.92
97.21 ± 11.01*
125.17 ± 12.63##
108.45 ± 12.17#
153.05 ± 14.57
113.49 ± 10.82*
141.72 ± 18.01##
120.12 ± 16.01#
8.83 ± 0.61
5.06 ± 0.64*
7.91 ± 0.52##
6.56 ± 0.45#
MTT reduction (lg formazon formed/min/mg protein)
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
Table 4 Effect of N-acetylcysteine (NAC) and Vitamin E administration on the activities of renal mitochondrial TCA cycle enzymes in EG ? NH4Cl treated (Hyperoxaluric) animals Control I
EG ? NH4Cl II
EG ? NH4Cl ? NAC III
Citrate synthase (n mol of DTNB reduced/min/mg protein)
16.51 ± 0.12
10.28 ± 0.39*
15.42 ± 0.63
##
12.24 ± 0.87#
Aconitase (n mol of cis Aconitate disappear/min/mg protein)
28.73 ± 1.16
17.42 ± 0.98*
25.21 ± 1.22
##
19.67 ± 1.64
Isocitrate dehydrogenase (n mol of NADPH formed/min/mg protein)
11.83 ± 1.46
6.79 ± 0.45*
9.95 ± 1.02
##
8.18 ± 1.12#
5.71 ± 0.89
2.07 ± 0.24*
4.90 ± 0.34
##
2.88 ± 0.38
Malate dehydrogenase (n mol of NADH oxidized/min/mg protein)
EG ? NH4Cl ? Vitamin E IV
Values are expressed as mean ± SD; n = 6 * Significantly different from control group (p \ 0.01) # ##
Significantly different from EG ? NH4Cl group (p \ 0.05) Significantly different from EG ? NH4Cl group (p \ 0.01)
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Mol Cell Biochem Fig. 4 Crystalluria depicted by polarization micrographs of experimental rat’s urine a Urine of control rats (group I) devoid of any crystals. b Urine of hyperoxaluric rats (group II) showing aggregate of COM crystals (Arrows indicate dumbbell-shaped COM crystals). c Urine of NACtreated rats (group III) showing few crystals. d Urine of Vitamin E treated rats (group IV) showing moderate amount of crystals. Original magnification 1009
Fig. 5 Renal histological analysis, stained with haematoxylin and eosin (original magnification 4009). a Control rats (group I). b Hyperoxaluric rats (group II). c NAC treated hyperoxaluric rats (group III). d Vitamin E treated hyperoxaluric rats (group IV). Arrows indicate Shrinkage of glomerulus and increased urinary space in hyperoxaluric rats
this study is supported by the presence of increased levels of MDA concentration in GSH depleted mitochondrial fractions in hyperoxaluric animals (group II). NAC significantly
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increased mitochondrial NP-SH (GSH) which might play a key role in the protection of mitochondrial components against hyperoxaluria-induced oxidative damage.
Mol Cell Biochem
We observed reduced activities of mitochondrial ETC complexes in hyperoxaluria, while NAC supplementation was observed to moderately reverse the loss. The decreased activities of mitochondrial enzymes in the present study are in agreement with the earlier studies in hyperoxaluric rats [13, 45]. Decrease in mitochondrial thiols following chronic hyperoxaluria might contribute to diminished activities of mitochondrial electron transport chain enzymes. It has also been shown that increased LPO or decrease in the antioxidants is associated with a loss of complex IV activity that consequently leads to mitochondria-dependent apoptosis [53]. Since kidney has a high energy demand, any decline in mitochondrial electron chain complex activities might have severe impact on kidney functions. Therefore, electron respiratory chain components appear to be the main mitochondrial targets of chronic hyperoxaluria. The restoration of mitochondrial enzyme activity by NAC might be the result of increase in the intracellular thiols which might offer protection by scavenging ROS or protecting thiols in mitochondrial proteins or both. The decrease in mitochondrial function in the present study was further evident from the diminished activities of the TCA cycle enzymes like citrate synthase, aconitase, ICDH, and MDH in hyperoxaluric animals. Increase in oxalate load might be the possible reason for the dwindling activities of these enzymes, as reactive oxygen species produced by oxalate/calcium oxalate can inactivate the mitochondrial enzymes [45]. NAC administration was able to reduce oxidative stress in hyperoxaluric mitochondria, this might be attributed to the normalization of activities of TCA enzymes. In conclusion, the present study confirms that mitochondrial oxidative stress and dysfunctions might contribute in part in the pathogenesis of hyperoxaluria-induced nephrolithiasis. In addition, the findings of the present study provide evidence that NAC might exert protective effects in hyperoxaluria through mitochondrial protection that may involve attenuation of oxidative stress. Further studies are needed in order to clarify the valid mechanism underlying these effects. Acknowledgments The financial assistance provided by the University Grants Commission, New Delhi is gratefully acknowledged. Conflict of interest
The authors state no conflict of interest.
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