Paracetamol or acetaminophen (N-acetyl-p-aminophenol; APAP) is a commonly used analgesic antipyretic drug and is used for an extensive range of treatments. APAP was reported to be safe and effective when used at therapeutic levels 1. However, an acute or cumulative overdose can cause APAP toxicity, which can produce hepatic and renal necrosis in both humans and experimental animals 2. Research results have raised the possibility that habitual acetaminophen use could increase the rate of progression of chronic renal disease 3. It was reported that, in rare circumstances, individuals died after taking less than the estimated minimum threshold toxic dose because of higher sensitivity to the toxic effects of APAP; hence, individual risk of toxicity following APAP overdose was difficult to assess 4.
It has been proposed that oxidative stress plays a significant role in APAP-induced toxicity 3. Therefore, drugs or plant products having antioxidant properties might have the potential to prevent APAP-induced nephrotoxicity 5.
Nigella sativa is a plant known to have antioxidant properties and its main component (constituting 30–48%) is thymoquinone, which has many therapeutic effects 6. The seeds of N. sativa are used extensively in the traditional medicine of many countries 7. Its oil extract (N. sativa oil; NSO) is most often used medicinally for its protective role against many diseases owing to the reported anti-inflammatory 8, antidiabetic 9, and hepatoprotective 10 activities. However, very few studies have dealt with its possible protective role against APAP-induced nephrotoxicity.
Aim of the work
The current study was designed to investigate the effect of APAP-induced toxicity on the structure of the rat renal cortex and assess the possible protective role of pretreatment with NSO using light and electron microscopic techniques in addition to estimation of plasma renin activity and arterial blood pressure.
Material and methods
Animals and treatments
Thirty adult male albino rats (3 months old; 250–300 g body weight) were obtained and maintained in the Animal Nutrition and Care House, Faculty of Medicine, Assiut University. The animals were treated in accordance with the published guidelines established by Assiut Council on laboratory Animal Care, and the experimental protocol was approved by the Institutional Animal Use Committee of Faculty of Medicine, Assiut University (Egypt). Animals were housed in properly ventilated cages with controlled temperature (25°C), humidity, and 12-h light/dark cycles and were allowed free access to rodent laboratory food and water throughout the experiment.
After 1 week of acclimatization, the rats were divided randomly into three groups of 10 animals each:
Group I: This group served as the control group and received the corresponding volume of APAP vehicle (saline).
Group II: This group was treated with 750 mg/kg/day APAP orally through a gastric tube for 7 consecutive days. This dose regimen of APAP was previously reported to induce renal damage in rats 11. APAP was purchased from Chemical Industries Development (CID) (Cairo, Egypt), and prepared as 20% suspension in saline stabilized by 0.2% gum.
Group III: The rats in this group received 2 ml/kg/day NSO orally 30 min before APAP oral administration at the same dose as for group II through a gastric tube for 7 consecutive days. The dose of NSO was chosen to be effective with no adverse effects as reported by Uz et al. 12. NSO was purchased from Kahira Pharmaceutical & Chemical Industries Co. (Cairo, Egypt), as 100% pure NSO.
The rats were administered the treatments in the morning after food supplementation to be sure that the stomach of the animals was full.
Twenty-four hours after the last dose of the experiment, arterial blood pressure was measured for each animal before decapitation. Kidney specimens and blood samples were obtained from each animal for histological and physiological studies.
After sacrifice, fresh small pieces were obtained from the kidney of each animal and fixed in 10% neutral formalin, cold acetone, and 4% glutaraldehyde.
Tissue specimens, fixed in 10% neutral formalin, were processed for preparation of paraffin sections (5-7 um) to be stained with Hematoxylin and Eosin stain (H&E) for studying the general histological structure and with Periodic acid Schiff method (PAS) for demonstration of neutral mucosubstances 13.
Tissue specimens, fixed in cold acetone, were processed to be stained by Calcium phosphate method for detection of alkaline phosphatase enzyme 13.
Kidney specimens, fixed in 4% glutaraldehyde, were processed for preparation of semithin sections (0.5–1 μm), stained with toluidine blue, and examined with a light microscope 14.
Ultrathin sections (500–800 A) from selected areas of semithin sections were contrasted with uranyl acetate and lead citrate 15, examined under a transmission electron microscope (Jeol – 100 CX; Japan), and photographed at 80 kV at the Electron Microscope Unit (Assuit University, Egypt).
Systolic blood pressure and diastolic blood pressure were measured in each rat using a pneumatic tail pulse transducer (Narco physiograph, model DMP4A; Biosystems Inc., Houston, Texas, USA).
Blood samples were obtained for measurement of plasma renin using an enzyme immunoassay with antirenin monoclonal antibodies.
Data, expressed as means±SEs, were subjected to analysis of variance (ANOVA) of a completely randomized design. Significance was ascertained using the Student t-test: P values less than or equal to 0.05 were considered significant. Computations were performed with SPSS version 8.1 (SAS Institute Inc., Cary, North Carolina, USA).
Light microscopic study
General histological examination of the kidney of control rats (group I) showed the renal cortex with a normal structure. Each renal corpuscle was formed of a glomerular tuft of capillaries surrounded by Bowman’s capsule, which had a parietal layer lined with simple squamous epithelium and a visceral layer formed of podocytes investing the glomerular capillaries. Intraglomerular mesangial cells were identified by their densely stained nuclei and were surrounded by a deeply stained matrix between the glomerular capillaries (Figs 1 and 2). The proximal convoluted tubules (PCTs) had rounded vesicular nuclei and highly stained cytoplasm with apical brush borders and basal striations. The distal convoluted tubules (DCTs) had apical rounded nuclei, pale stained cytoplasm, and a wider lumen (Figs 1 and 3). The juxtaglomerular apparatus consisted of juxtaglomerular cells, macula densa cells, and extraglomerular mesangial cells. The juxtaglomerular cells (renin-secreting cells) were modified smooth muscle cells located in the wall of afferent arterioles in its terminal portion at the transition into the glomerular capillary network and contained groups of densely stained renin granules in their cytoplasm. Macula densa cells were cells of the adjacent DCTs in close proximity to the renal corpuscle that appeared crowded with closely packed nuclei and were separated from juxtaglomerular cells by extraglomerular mesangial cells that appeared similar to intraglomerular mesangial cells (Fig. 2).
The renal cortex of APAP-treated rats (group II) revealed marked necrotic changes in PCTs that exhibited pyknotic densely stained nuclei and a vacuolated cytoplasm with loss of integrity of their brush border and loss of basal striations. Some pyknotic nuclei were observed in the lumen of PCTs. The juxtaglomerular cells showed fewer dense renin granules compared with the control group. Mild vascular congestion was observed. The structure of DCTs and the collecting tubules was relatively preserved (Figs 4–6).
Sections of the renal cortex of NSO-treated and APAP-treated rats (group III) showed preservation of a near to normal structure for many (PCTs) with regular brush border and well-organized basal striations. A few tubular cells exhibited pyknotic nuclei and vacuolated cytoplasm. A noticeable increase in dense renin granules was noticed in the juxtaglomerular cells compared with group II but they were still fewer than in the control group. Congestion of some glomerular and peritubular capillaries was also noticed (Figs 7–9).
PAS-stained sections of the renal cortex of control rats (group I) revealed a PAS-positive reaction along the basement membranes of the renal tubules, the parietal layer of Bowman’s capsule and glomerular capillaries, and along the apical brush border of the PCT (Fig. 10). Sections of the renal cortex of APAP-treated rats (group II) showed detachment of many tubular epithelial cells from their basement membrane and noticeable interruption in the PAS-positive reaction along the brush border of many PCTs (Fig. 11). In the renal cortex of NSO and APAP treated rats (group III), the tubular epithelial cells appeared to be resting on their PAS-positive basement membrane and a PAS-positive reaction was noticed along the uninterrupted brush border of most PCTs (Fig. 12).
Examination of kidney sections stained for alkaline phosphatase enzyme revealed an intense positive reaction for alkaline phosphatase in the apical brush border of PCTs in the control rat renal cortex of group I (Fig. 13). The renal cortex of APAP-treated rats (group II) showed a noticeable reduction in alkaline phosphatase-positive reaction in the interrupted brush border of some PCTs (Fig. 14). Sections of the renal cortex of NSO and APAP treated (group III) revealed increased reaction for alkaline phosphatase along the continuous brush border of many PCTs compared with group II but it was still lower than that of control animals (Fig. 15).
Electron microscopic study
In control rats, podocytes had a folded nucleus and cytoplasm containing bundles of filaments, free ribosomes, scattered strands of rough endoplasmic reticulum, and a few mitochondria. Their cell body gave rise to primary processes and numerous secondary processes (foot processes) separated by filtration slits of uniform width and resting on the glomerular basement membrane (GBM). The GBM exhibited a central electron-dense lamina densa bordered on each side by an electron-lucent lamina rara. The glomerular capillaries were lined with fenestrated endothelium (Fig. 16). PCTs cells showed numerous long apical microvilli and well-developed basal infoldings containing numerous elongated mitochondria in addition to some apical vacuoles immediately beneath the microvilli and a few lysosomes in the cytoplasm. Their nucleus was euchromatic with a prominent nucleolus (Fig. 17). The cells of DCTs had apical euchromatic nuclei, well-developed basal infoldings enclosing elongated mitochondria, a few vacuoles, a few lysosomes, and a few luminal microvilli (Fig. 18). The tubular basement membrane of PCTs and DCTs appeared regular and thin (Figs 17 and 18). The juxtaglomerular cells characteristically contained aggregates of electron-dense renin granules of variable sizes and shapes in their cytoplasm. Macula densa cells in the adjacent DCTs were characterized by closely packed nuclei and dispersed mitochondria that had little association with the reduced infoldings of the basal membrane (Fig. 19).
The renal cortex of the APAP-treated group showed focal fusion and flattening of secondary foot processes related to some glomerular capillaries and irregular thickening of the GBM with partial loss of its trilamellar structure in some renal corpuscles (Fig. 20). Obvious degenerative changes were noticed in many PCTs. Some PCT cells revealed a noticeable reduction and distortion of microvilli with increased electron density of their nuclei. Their cytoplasm showed many peroxisomes, which appeared as membrane-limited bodies with an electron-dense inclusion suspended in a homogenous matrix of lower electron density (Fig. 21). Other PCT cells showed reduced basal infoldings, loss of longitudinal organization of the mitochondria, which changed from elongated to almost circular in shape with an uneven distribution in the cytoplasm, many lysosomes of variable sizes, large lipid droplets, and apical displacement of nuclei (Fig. 22). With respect to DCTs, there was a relative preservation of the ultrastructure of their lining cells. Some distal tubular cells showed a disarrangement of the mitochondria (Fig. 23). The tubular basement membrane of some PCTs and DCTs appeared irregular and thick compared with that of the control group (Figs 22 and 23). Most juxtaglomerular cells showed few electron-dense renin secretory granules in their cytoplasm compared with control rats (Fig. 24).
The ultrastructure of the renal cortex of rats treated with NSO and APAP revealed a relatively normal arrangement of secondary foot processes and regular thickening of the GBM with restoration of its trilamellar structure along most glomerular capillaries (Fig. 25). Many PCT cells showed intact apical microvilli, basal infoldings containing numerous elongated mitochondria, a few lysosomes and lipid droplets, and euchromatic basal nuclei (Fig. 26). Most DCT cells showed a relatively regular arrangement of mitochondria (Fig. 27). Many PCTs and DCTs showed a regular tubular basement membrane of their lining cells (Figs 26 and 27). Examination of juxtaglomerular cells revealed a considerable number of electron-dense renin granules of variable shapes and sizes in the cytoplasm (Fig. 28).
It has been shown that APAP administration significantly increases systolic blood pressure, diastolic blood pressure, and plasma renin activity. It was interesting that the alterations that took place after APAP administration were partially normalized after administration of NSO (Fig. 29).
The results of the current work demonstrated that APAP overdose induced marked structural changes in the rat renal cortex that mainly involved the PCTs with some affection of the renal corpuscles, DCT, and juxtaglomerular cells.
The present study revealed marked affection of PCTs in APAP-treated rats. PCT cells showed degenerative changes in the form of dense nuclear staining, cytoplasmic vacuolization, loss of integrity of the apical brush border, partial loss of basal striations, disorganization of mitochondria, and increased peroxisomes, lysosomes, and some lipid droplets with irregularity of their basement membrane. This was in accordance with previous studies that reported that APAP overdose produced acute proximal tubular necrosis in male rats 16.
The most remarkable structural change in PCT cells was the loss of integrity and distortion of their brush border, which was evidenced by reduced PAS and alkaline phosphatase reaction and by marked disruption of apical microvilli in ultrathin sections. Damage to PCT cells could result in increased urinary excretion of the key brush border membrane enzyme, alkaline phosphatase 17. This was in agreement with the study by Trumper et al. 16, who reported that APAP promoted a reduction in alkaline phosphatase activity in the brush border membrane and an increase in the urinary excretion of the brush border membrane enzyme γ-glutamyl transpeptidase 16.
The partial loss of the basal striations observed in the present work probably occurred as a result of damage to the cytoskeleton of PCT cells resulting in decreased activities of the basolateral membrane enzyme Na+, K+, ATPase 18. Normally, PCTs reabsorb two-thirds of the Na+ and H2O filtered by the glomerulus 18. The polar distribution of Na+, K+, ATPase to the basolateral membrane is essential for efficient Na+ reabsorption 16. Therefore, APAP-induced loss of basal striations impaired Na+ and H2O reabsorption, which, together with disruption of the brush border, could account for the reported elevation of Na+ and H2O urinary excretion 19.
The mild affection of mitochondria observed in our study was in accordance with that seen in previous studies, which reported that APAP-induced renal alterations occurred without changes in cortical ATP content, suggesting that the observed effects of APAP were not mediated by a depletion of cellular energy stores 16 with possible involvement of other biochemical events such as disruption of calcium homeostasis 20.
In the present work, numerous peroxisomes were observed in many PCT cells. Peroxisomes are cytoplasmic organelles known to be rich in catalase, which is involved in the destruction of hydrogen peroxide 21; therefore, increased incidence of peroxisomes may be because of increased toxic oxidative insults in PCT cells. In accordance with this, an increase in the number of peroxisomes (peroxisomal proliferation) was a striking feature in rat renal tubular cell injury 22. Further, our findings showed a noticeable increase in lysosomes and the appearance of lipid droplets in PCT cells, which were in accordance with the findings of Ucheya and Igweh 4, which revealed droplets in the renal tubules of pregnant rats with long-term APAP administration, indicating the presence of toxic cellular insults that may be because of accumulating reactive oxygen species (ROS) or abnormally filtered substances absorbed by tubular epithelial cells 23. Lysosomes when exposed to toxic insults increase in size and number and undergo phospholipidosis 24. It has been reported that cytoplasmic accumulation of lysosomes indicates cell injury 25.
The observed irregularity and thickening of the tubular basement membrane could be a compensatory response to the disrupted brush border of PCTs, along which many substances were absorbed. Similarly, studies dealing with the renal lesions of analgesic nephropathy found increased thickness of tubular and capillary basement membranes 26.
In the present study, some desquamated cells were observed in the PCT lumen that may be because of cytoskeletal alterations and impaired cell-substratum adhesion in PCT cells of APAP-treated rats. These desquamated cells may be the cause for granular casts and epithelial cells being detected in the urinary sediments of APAP-treated rats 27.
The present work demonstrated some degenerative changes in the renal corpuscles involving irregular thickening of the GBM in addition to fusion and flattening of secondary podocyte processes. Thickened GBM was a common finding of many pathologic and experimental conditions 20,28, leading to increased permeability of glomerular capillaries resulting in proteinuria. Damage to the GBM itself was attributed to ROS attacking the GBM and damaging its intricate matrix structure 29. This may occur by direct oxidation of the GBM components or by adduct formation and dimerization of type IV collagen, leading to distortion of the GBM 30. Degradation of the matrix structure of the GBM can explain the resulting proteinuria 28. Fusion and flattening of foot processes were reported to result from direct injury to the podocyte skeleton after exposure to ROS 31 or may develop secondary to the thickened GBM to compensate for the increased glomerular permeability and proteinuria 17.
In the present study, DCT appeared much less affected than PCT, which suggested that APAP nephrotoxicity may affect DCT secondary to changes in PCT.
The tubular and glomerular injury observed in the current study was in agreement with the renal cortical damage reported in previous studies 3,32 and explained the impaired renal functions with APAP treatment observed in previous works 16,33. In contrast to our results, Nassar et al. 34 reported that there were no detectable histological changes in kidney tubule cells of mice treated with APAP. This controversy may be attributed to the different doses and periods of administration of APAP in this study.
A striking feature in the present study was the noticeable reduction in the renin granule pool in the cytoplasm of juxtaglomerular cells of APAP-treated rats, which coincided with increased plasma renin activity and significant increase in arterial blood pressure. Juxtaglomerular cells have been known to synthesize and store renin in mature secretory granules and release these granules by exocytosis 35. The rate of renin granule exocytosis determined the level of activation of the renin–angiotensin–aldosterone system such that the reduced number of renin granules after acute stimulation of juxtaglomerular cells was accompanied by increase in plasma renin activity 36. Renin secretion displayed a uniquely high sensitivity to changes in extracellular osmolality so that low epithelial Na+ Cl− transport across the renal tubules induced a reduction in extracellular osmolality, leading to a swelling of juxtaglomerular cells and increase in renin secretion 37. Therefore, the observed APAP-mediated stimulatory effect on renin secretion in the present study may be because of the direct stimulatory effect on juxtaglomerular cells or because of an indirect effect related to low epithelial tubular transport of Na+ Cl−. To our knowledge, very little has been published about the effect of APAP on the structure of juxtaglomerular cells in the literature. Panos et al. 38 stated that plasma levels of renin and aldosterone were increased in patients with APAP fulminant hepatic failure. In contrast, Sudano et al. 39 reported that, in patients with coronary artery disease treated with APAP, the plasma renin activity remained unchanged. The noticeable increase in blood pressure, in the present work, could be attributed to the evident renal structural and functional impairment in addition to increased renin secretion with subsequent stimulation of the renin–angiotensin system. Substantial evidence implicated impaired renal excretion of Na+ as the major factor in the pathogenesis of hypertension 40. In support, Forman et al. 41 found that men who took acetaminophen 6–7 days/week demonstrated an increased relative risk for incident hypertension.
There was accumulating evidence that ROS and free radicals were important mediators of APAP toxicity 3. The first demonstration of its toxicity was the formation of the reactive intermediate N-acetyl-p-benzoquinone imine by cytochrome P450, which, at therapeutic doses, was removed by conjugation with glutathione (GSH) sulfhydryl. High doses of APAP resulted in the depletion of cellular GSH, which allowed N-acetyl-p-benzoquinone imine to bind to cellular proteins and initiate lipid peroxidation and cell death leading to renal injury 42. APAP-induced renal injury could also be because of hepatic-derived APAP metabolites, particularly GSH conjugates 16. Moreover, the inhibitory effect of APAP on renal PG synthesis could result in impaired renal function with a reduced glomerular filtration rate and a decreased effective renal plasma flow 43.
The present study declared that the deleterious effects of APAP on the renal structure were noticeably ameliorated by concomitant supplementation with NSO. To a great extent, a relatively normal structure of the renal glomeruli, PCT, DCT, and juxtaglomerular cells was retained. Examination of PAS and alkaline phosphatase reaction confirmed this observation. Also, the plasma renin activity and arterial blood pressure were obviously reduced to near normal values. However, there were still some tubular cells with dense nuclei and vacuolated cytoplasm in addition to noticeable vascular congestion. NSO was proved to have strong antioxidant properties and most of its pharmacological actions were reported to be because of its ability to scavenge free radicals and inhibit lipid peroxidation 44 in addition to its pivotal role in protecting the renal tissue against severe hazardous effects of many agents such as cyclosporine 12, gentamicin 45, and cisplatin 46. Thymoquinone, the main constituent of NSO, was reported to be effective in protecting mice against APAP-induced hepatotoxicity possibly by increasing resistance to oxidative stress and improving mitochondrial function 6.
In conclusion, NSO was seen to protect the renal architecture against hazardous effects of APAP overdose, which primarily involved PCT. Oxidative stress is one of the mechanisms by which APAP causes renal damage, and NSO, through its antioxidant properties, represents a promising therapeutic agent that protects renal tissue. The extent of APAP abuse, as a major community health problem, should be carefully researched. Restricted APAP prescription and careful follow-up of renal function during its consumption should be carried out in patients with renal disease. Further studies should investigate the effect of therapeutic doses of APAP for variable durations.
The authors thank Dr Omyma Galal Ahmed, Assistant Professor of Medical Physiology, Medical Physiology Department, Faculty of Medicine, Assiut University, Egypt, for her kind help and support in physiological assessments.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2013 The Egyptian Journal of Histology
Nigella sativa oil; paracetamol; renal cortex