Histopathological, Immunohistochemical and Biochemical …curresweb.com/mejas/mejas/2017/870-887.pdf · 2017-12-05 · affects by toxic dose of APAP after liver is kidney (Bertolini

Middle East Journal of Applied Sciences ISSN 2077-4613

Volume : 07 | Issue :04 |Oct.-Dec.| 2017 Pages: 870-887

Corresponding Author: G. El-Sherif, Department of Zoology & Entomology, Faculty of Science, Minia University, Minia, Egypt. E-mail: [email protected]

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Histopathological, Immunohistochemical and Biochemical Studies on the Effects of Paracetamol and Green Tea Extract on Male Albino Rat Kidney

El-Sherif G., Hanan A. El-Bakry and Rehab M. Rostom Department of Zoology & Entomology, Faculty of Science, Minia University, Minia, Egypt.

Received: 03 August 2017 / Accepted: 05 Sept. 2017 / Publication date: 23 Nov. 2017 ABSTRACT

Paracetamol (acetaminophen) is a pain reliever and a fever reducer often used to treat many conditions such as headache, muscle aches, arthritis, backache, toothaches, colds, and fevers. However, Green tea extracts have been widely used as food supplements which are believed to have antioxidant, anticarcinogen, anti-inflammatory, and anti-radiation biochemical effects. It was found that administration of 400 mg/200 g paracetamol exhibited many histopathological lesions in all renal tissues. On the other hand, administration of 1.7 mg/200 g of green tea extract resulted in variable histopathological effects. On applying paracetamol followed by the green tea extract, some lesions increased, while others appeared. Leaving experimental animals to recover for one month after administration did not exhibit any healing mechanisms regarding degeneration, necrosis and apoptosis of renal tissue cells but does not affect other lesions. Collagen fibers, PAS reaction and MDA (malonyldialdehyde) increased in all experimental groups. As different findings, GSH (Glutathione) and catalase activity decreased while caspase-3 reaction was found positive in all experimental groups. These results were discussed, correlated and evaluated on the bases of effects of paracetamol and green tea extracts on rat renal tissues either as a single or as a dose followed an administrated doses. It was concluded that the histopathological and immunohistochemical investigations of renal tissues confirmed the biochemical assessment results which explicate the incidence of severe renal damage in rats treated with paracetamol, green tea extract and paracetamol followed by green tea extract in addition to rats left to recover. Key words: Paracetamol, Green Tea Extract, Catalase, Masson's Trichrome Stain , Caspase-3 , Rat

Kidney

Introduction

Paracetamol (N-acetyl-p-aminophenol; APAP) is also known as acetaminophen, is a broadly consumed analgesic and antipyretic agent, which is safe and well tolerated when used in the normal therapeutic dosage. Nevertheless, overdose of APAP is accompanied with hepatic and renal damage in both human and experimental animals (Refaat and Mady, 2008). The second target organ that affects by toxic dose of APAP after liver is kidney (Bertolini et al, 2006). Renal injury and acute renal failure can arise even in the absence of liver injury (Bertolini et al, 2006). Renal insufficiency happens in nearly 1–2% of the patients with a high dose of APAP (Perscott, 1983). Recent evidences recommended that oxidative stress with increased development of reactive oxygen species, diminution of reduced glutathione (GSH) and lipid peroxidation play a critical role in the generation of paracetamol-induced renal injury(Liu et al, 2011).

Kidneys are the main excretory organs of the body; they accomplish the crucial task of filtering the blood from toxic materials, such as toxic medications, poisonous chemical substances and toxic metabolites, by liberating them out of the blood and excreting them in the form of urine (Bhattacharya and Bhakta, 2016).

Natural herbs are commonly taken by humans on a daily basis; these natural products have several biologic and pharmacologic properties (Hosseinimehr, 2014). Herbs are naturally rich in bioactive plant products with food value to preserve energy balance in the body and significant curative value in several diseases (Sharma, 2010). Majority of the public considered herbal medicine as having low side effects and can improve the consequences of conventional agents or be a substitute

Middle East J. Appl. Sci., 7(4): 870-887, 2017 ISSN 2077-4613

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treatment (Desai and Grosberg, 2003). Among these herbs is the green tea, which is widely consumed due to its health-promoting potential. Green tea (Camellia sinensis), is the most popular beverage consumed in the world next to water. It belongs to the family Theaceae (Bhattacharya and Bhakta, 2016). It has been shown to have cardioprotective, neuroprotective, antidiabetic, and antimicrobial properties (Imai and Mukhtar, 1997; Ahmad and Mukhtar, 1999; & Khan and Mukhtar, 2007). Moreover, green tea has been illustrated to ameliorate kidney function in diabetic rats (Rhee et al, 2002). Also, It has been established to improve cyclosporine A and cisplatin-induced renal dysfunction in experimental rats (Mohamadin et al, 2005 & Patil and Balaraman, 2005).

Green tea is a non-fermented tea and encompasses more catechins than black tea, its content of definite minerals and vitamins elevates the antioxidant potential of this kind of tea. Green tea comprises approximately 4000 bioactive ingredients of which one third is contributed by polyphenols (Mahmood et al.,2010), other compounds are alkaloids (Caffeine, theophylline, chlorophyll, fluoride, aluminum, minerals and trace elements) (Cabrera et al, 2003). Polyphenols present in green tea are mainly flavonoids, the Polyphenols are a large group of plant chemicals that contain catechins are thought to be responsible for the health benefits that have traditionally been attributed to tea especially green tea (Babu et al, 2006). The most important catechins found in green tea are epicatechingallate (ECG), epicatechin(EC), epigallocatechin (EGC) and epigallocatechingallate (EGCG) (Wu and Yu, 2006).

Despite the favorable data reinforcing the benefits of a diet rich in green tea and its accompanied polyphenols, some researchers (Chang et al, 2003; Stratton et al, 2000 & Isbrucker et al, 2006) have studied their potential toxicity when taken at high doses, as a concentrated product, or as a nearly-pure compound free from interactions with other chemicals normally found in green tea extract. Aim of the work:

In the present study, effects of using paracetamol, to induce hepatotoxicity and green tea extracts, as dietary supplements, on renal tissues have been evaluated using specific reliable histopathological, immunohistochemical and biochemical techniques. The dual effects of using paracetamol followed by green tea extract have also been studied, in addition to studying rats left to autorecovery as well. Materials and Methods Drugs:

Paracetamol (APAP) was purchased as a commercial drug [Novaldol 1000mg] from Sanofi-Aventis Company, Egypt s.a.e., El-Sawah, El-Amiria, Cairo. Each tablet contains 1gm (1000 mg) paracetamol (in the form of paracetamol DC 90). Green tea extract (GTE) tablets were obtained as a dietary supplement (which is commercially known as Multi–treat) from the Arab Co. for Pharmaceutical & Medicinal plants (MEPACO-MEDIFOOD) at Enshas, Ei-sharqeia Governorate, Egypt. Each tablet contains 300 mg green tea dry extract (30% polyphenols).

Animals:

Male albino rats (Rattus norvegicus) weighing about 225-250 gm, were obtained from the breeding colony of the Ministry of Health (Helwan, Egypt). All animals were housed in plastic cages and kept under the same laboratory conditions of temperature (25±2°C) and lighting (12:12hrs light: dark cycle). Rats were orally provided ad libitum with water and fed with standard commercial rat chow. Animal procedures were performed in accordance with institutional guidelines and follow the Guide for the Care and Use of Laboratory Animals (2011).

Experimental design:

After two weeks of acclimatization, male rats were randomly divided into six groups (9 animals in each) and administered as follows:


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A. Group I: was provided with normal diet and water ad libitum daily and kept as control); B. Group II: was treated orally with paracetamol (400 mg/200 g /day) for one week; C. Group III: was treated orally with green tea extract (1.7 mg/200g/day) for one month; D. Group IV: was treated orally with paracetamol (400 mg/200 g /day) for one week followed

by green tea extract (1.7 mg/ 200g/day) for one month; E. Group V: was treated orally with paracetamol (400 mg/200 g /day) for one week and then left

to recover for one month and F. Group VI: was treated orally with paracetamol (400 mg/200 g /day) for one week followed by

green tea extract (1.7 mg/ 200g/day) for one month and left to recover for one month.

Table 1: Experimental design

Ser.

Group

Drugs Duration

Recovery Paracetamol,

400 mg/200 g Green Tea, 1.7 mg/ 200 g

1 I 9 ♂ -----------------------

----------------------

--------------------

-------------------

2 II

9 ♂ Paracetamol, 400 mg/200 g

----------------------

One week

------------------

3 III

9 ♂ -----------------------

Green Tea, 1.7 mg/ 200 g

One month

------------------

4 IV



APAP. One week GTE One month

------------------

5 V 9 ♂ Paracetamol, 400 mg/200 g

----------------------

One week

One month

6 VI



APAP. One week GTE One month

One month

Collection of samples:

Animals sacrificed under deep anaesthesia with ether; two portions of renal tissues were harvested; the first portion was fixed in 10% formalin for histopathological and immunohistochemical studies and the other portion was immediately stored at -80οC for measurement of oxidative stress and antioxidant parameters.

Experimental procedures: (A) Biochemical studies: Measurement of Lipid Peroxide (measured as MDA): Free malonyldialdehyde (MDA) in the kidney (as a reactive, potentially mutagenic and a marker for the oxidative stress, Hartman, 1983) was determined colorimetrically using a commercially-available kit (Bio-Diagnostic, Egypt) according to the method of Ohkawa et al, 1979. Measurement of Glutathione (GSH): GSH level (as an antioxidant preventing damage to important cellular components, Pompella et al, 2003) was determined colorimetrically in the homogenates of kidney using commercial kit (Bio-Diagnostic, Egypt) following the method of Beutler et al, 1963. Measurement of Catalase Activity: The activity of catalase (as an enzyme protecting the cell from oxidative damage, Goodsell, 2004) was measured colorimetrically in the homogenate of kidney using catalase assay kits (Bio-Diagnostic, Egypt) as described by Aebi, 1984.


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Histopathological studies: Formalin-fixed hepatic and renal tissues were dehydrated, embedded in Paraplast, cut at 5 μm and stained with haematoxylin and eosin (H & E) to verify histological details, Masson’s trichrome to demonstrate the collagen fibers and Periodic Acid Schiffs (PAS) staining for detecting polysaccharides. Immunohistochemical Studies: Immunohistochemical staining with immunolabeling for activated caspase-3 for kidney sections, (5 μm) was performed (Krajewska et al, 1997). Statistical analysis: Statistical analysis was performed using ANOVA (SPSS software, version 16.0, SPSS Inc. Chicago, IL, USA). Post-Hoc comparisons of pair-wise means were made using Least Significant Difference (LSD) test. The results were presented as mean ± standard error of the mean (SEM). Differences between group means were considered statistically significant if P-value <0.05. Results Biochemical studies: Malonyldialdehyde (MDA):

Figure (1) demonstrated that MDA level of renal tissues exhibited a significant elevation (p

<0.05) in animals of all treated groups when compared with control group. Likewise, there was a significant increase (p <0.05) in the level of renal MDA in animals of all other treated groups (III, IV, V and VI) when compared with paracetamol treated group (II) but results of groups IV and VI are still recording the maximum values of MDA levels.

Fig. 1: Effects of APAP and GTE on renal MDA of male rats

Glutathione (GSH): Results found in Figure (2) revealed that there was a significant reduction (p <0.05) in renal

GSH in animals of all treated groups (II - VI) when compared to the control group.


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Fig. 2: Effects of APAP and GTE on renal GSH of male rats

Regarding GSH, there was non-significant difference (p >0.05) between paracetamol treated group (II) and other treated groups.

Catalase activity:

As illustrated in Figure (3), the activity of catalase in kidney was significantly declined (p <0.05)

in animals of all treated groups when compared to control group. Furthermore, there was a significant decrease (p <0.05) in catalase activity in kidney of paracetamol followed by green tea extract group (IV) and paracetamol followed by green tea extract recovery group (VI) when compared with paracetamol group (II). While non-significant difference (p >0.05) was observed between green tea extract group (III), paracetamol recovery group (V) and paracetamol group (II).

Fig. 3: Effects of APAP and GTE on renal Catalase of male rats Histopathological studies: H & E staining:

Examination of kidney of control rats (Group I) showed normal architecture of renal tissue

which is classically composed of cortex and medulla. The cortical tissue consists of cortical tubules and renal corpuscles; the renal corpuscles appear as dense rounded structures comprising of glomeruli surrounded by a double-walled cup (Bowman’s capsule) lined by squamous epithelial cells (Fig. 4).


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The cortical tubules represent the main bulk of the cortical parenchyma and mainly consist of proximal and distal convoluted tubules. Proximal convoluted tubules are lined by simple cuboidal epithelium with prominent brush border, and each proximal tubule cell contains a single spherical nucleus. Distal convoluted tubules are also lined with simple cuboidal epithelium but had more clearly defined lumen and without brush border (Fig. 4).

The medullary tissues contain thin descending limb of loop of Henle (straight portion of proximal tubules) lined by simple squamous epithelium, and thick ascending limb of loop of Henle (straight portion of distal tubules) line by simple cuboidal epithelium. It also contain collecting tubules which are lined by simple cuboidal epithelium but slightly wider and less regular in shape than the thick ascending limb of loop of Henle (Fig. 5), in addition to the collecting ducts or papillary ducts which are recognized by columnar epithelium.

Kidney of paracetamol treated rats (Group II) showed altered cortical architecture manifested by the presence of a marked ischemic collapse as the renal corpuscle appear smaller in size (Fig. 6), accompanied by degeneration of other glomeruli (Fig. 7) with hypertrophy in some renal corpuscles (Fig. 8). In addition, kidney sections of medullary tissues of paracetamol-treated rats showed loss of brush border of proximal convoluted tubules, the occurrence of multiple focal tubulo-nephritis with marked lymphocytic infiltrations (Figs. 9 & 10), the presence of vacuolated cells with pyknotic nuclei as well (Fig. 10), and the appearance of protein casts with squamation of renal tubular cells (Fig. 11). Also, kidney of paracetamol treated animals (Group II) exhibited necrosis and a cystic space (Fig. 12) and apoptotic cells with interstitial congestion (Fig. 13) in cells of proximal and distal tubules.

Kidney of green tea extract treated rats (Group III) showed extensive similar alteration of glomeruli and tubular structures as that recorded in paracetamol treated animals (Group II), which exhibited the same features of ischemic collapse, mesangial hyper cellularity and degeneration of glomruli in some renal corpuscles. Furthermore, interstitial congestion, necrosis, presence of cystic spaces and squamation of cells in renal tubules and apoptosis (Fig. 14) in cells of proximal and distal tubules were also noticed.

The renal tissues of rats treated with paracetamol followed by green tea extract (Group IV) revealed almost similar histopathological architecture if correlated with that were found in group II and group III in addition to marked vaculated cells & pyknotic nuclei (Fig. 15).

In rats left to recover for 1 month after paracetamol administration (Group V), kidney tissues exhibited just weak improvement in tubular structure with a little bit glomerular damage. In recovery group given paracetamol followed by green tea extract (Group VI), kidney showed decreased number of degenerative glumerulai with lesser tubular damages. Nevertheless, other histopathological lesions described in other experimental groups were still recorded.

PAS staining for carbohydrate content:

The renal tissues of the control animals (group I) showed PAS positive staining in the renal

glomeruli, brush borders of proximal tubules and in the basement membranes of proximal and distal tubules (Fig. 16)

If compared to group I, examination of renal tissues of group II animals, that were treated with paracetamol showed an intense PAS positive staining in the glomerular capillary basement membranes (GBMs) which appear more thickened and indicating the occurrence of membranous glomerulonephritis which is noticed in the renal tubules as well (Fig. 17).

In animals that were treated with green tea extract (group III), renal tissues showed more intense PAS positive staining demonstrated mainly in the renal tubules and the (GBMs). In addition, an increase in polysaccharide content among the renal tubules was also found (Fig. 18)

In group IV animals, that were treated with paracetamol followed by green tea extract, PAS positivity was the same as that in group II and group III in both glomeruli and also among renal tubules.

In recovery groups (V & VI), there was a similar increased PAS positive staining especially in glomerulus (thick arrows) and in tubular cells (Fig. 19).


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Masson's trichrome stain: The renal tissues of the control group I showed normal appearance of collagen fibers both in the

glomeruli and interstitial tissues among renal tubules (Fig. 20) In group II the collagen was highly increased which is manifested by accumulation of fibrous

tissues in the glomeruli (Fig. 21) and interstitial tissues (Fig. 22) In group III, and if compared to group II, there were more increase in collagen fibers in the renal

corpuscles (Fig. 23) and in the interstitium among the renal tubules (Fig. 24). There was a massive accumulation of fibrous tissues in the glomeruli and also in the interstitial

tissues among the renal tubules of group IV. In recovery groups V & VI, (Figs. 25) there was a weak response to recovery, as the collagen fibers were

still more increased than that in group I and like that in groups II, III and IV. Immunohistochemical studies:

Immunohistochemical staining of renal tissues of the control group rats (Group I) showed a

negative immunolabeling for activated caspase-3 in renal tubules and glomeruli (Fig. 26). The renal tissues of paracetamol treated group (Group II) showed high caspase-3 expression in

the glomeruli and renal tubules (Fig. 27). The expression of caspase-3 in renal tissue of green tea extract treated group (Group III) was

similar to that expressed in group II in both glomeruli (Fig. 28) and renal tubules as well. The renal tissues of rats treated with paracetamol followed by green tea extract (Group IV)

showed an extensive positive reaction to caspase-3 in renal tubules and glomeruli (Fig. 29). Considering the recovery improvement expectations, the expression of caspase-3 in renal tissues

of recovery groups (Group V & VI) is still very high (Figs. 30 & 31). Discussion Biochemical studies: Malonyldialdehyde (MDA), Glutathione (GSH) & Catalase activities:

In the current study, paracetamol administration exhibited some significant biochemical changes

in renal tissues varied among depletion in GSH content, a reduction in catalase activity and an increase in lipid peroxidation, which are in accordance with Hamza and Al-Harbi (2015). They reported that treatment with paracetamol resulted in a significant enhance in MDA with simultaneous decrease in catalase activity and GSH stores in mice kidney. Moreover, the results of this study are in agreement with Yousef et al (2010), since they reported that paracetamol administration led to a significant elevation in MDA levels accompanied with inhibition in the activities of antioxidant enzymes and catalase in rat plasma, liver, kidney, brain, lung, heart and testis. Furthermore, it reduce glutathione store significantly in rat liver, kidney and lung.

The results of the current study are reinforced with Hassan and A. Rahman (2016) were found that paracetamol stimulate a rapid loss in GSH and catalase and increase in lipid peroxidation in liver, kidney and brain. Similar results are obtained by Abd El Kader and Mohamed (2012) as they observed that treatment with paracetamol caused significant decrease in GSH and catalase content in addition to significant increase in MDA level in rat liver and kidney.

Both paracetamol and its reactive metabolite NAPQI (N-acetyl-p-benzoquinone imine) can interact with mitochondria, thus triggering reduction of mitochondrial GSH content; diminish in ATP content, and uncoupling of the mitochondrial respiratory chain combined with electron leakage (Donnelly et al, 1994).

Extreme ROS generation by NAPQI stimulates an imbalance of oxidative–antioxidative processes and results in lipid peroxidation and antioxidant decline. The reduction of catalase may be due to futile cycling of P450 (the spectrophotometric peak at the wavelength of the absorption maximum of the enzyme (450 nm) caused by NAPQI, which consumed Nicotinamide adenine dinucleotide phosphate (NADPH), associated with reduction of molecular superoxide radical (Uzkeser et al, 2012).


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Similarly, in this study, green tea extract caused significant elevation in MDA levels and reduction in GSH content as well as catalase activity in rat kidney.

In contrast, Gad and Zaghloul (2013) documented that treatment of rats with green tea extract lead to significant increase in GSH and significant reduction in MDA level in both liver and kidney. Histopathological studies: H &E staining:

In the current study, treatment with high dose of paracetamol (400mg/ 200g) induced several

histopathological lesions in renal tissues which represented by extensive destruction of glomeruli, multiple focal tubulonephritis with marked lymphocytic infiltrations, vaculated, apoptotic and necrotic cells of the proximal and distal tubules, appearance of protein casts as well as squamation of cells of renal tubules. Similar observations have been reported by Refaat and Mady (2008). Moreover, Adeneye and Olagunju (2009) observed that administration of paracetamol led to multiple focal tubulonephritis characterized by oedematous tubular cells with vacuolar degeneration and marked lymphocytic infiltration. Some authors, as Dhibi et al, (2014), emphasized the presence of marked alterations in the glomeruli and the proximal tubules. Furthermore, the histopathological results of the present study are reinforced by Cekmen et al, (2009), they noted that paracetamol treated rats suffer from various changes in the renal tissue involved tubular epithelial degeneration, vacuolization, cell desquamation and necrosis.

It is well established that paracetamol act via inhibition of prostaglandin synthesis (Harvey et al, 2009) and kidney is very active in the production and metabolism of prostaglandins. These compounds are contributed in numerous physiological processes in the kidney comprising auto-regulation of renal blood flow and glomerular filtration, modulation of renin release, tubular ion transport and water metabolism (Gross et al, 1981). Yasmeen et al. (2007) reported that depression of prostaglandin in renal tissues led to constriction in renal arterioles which then caused ischemia and finally enhanced into tubular epithelial cell injury. They also stated that, in response to ischemia or many toxins, histological injuries induced rapid loss of cytoskeletal integrity and cell polarity. However, our findings regarding the flaking of proximal tubule brush border, loss of polarity with mis-localization of adhesion molecules and other membrane proteins such as Na +, K +-ATPase and β-integrins were also established as mentioned by Zuk et al, 1998.

It was investigated that cell death was attributed to apoptosis and necrosis (Thadhani et al, 1996), with serious damage to both viable and nonviable cells that are then desquamated, leaving areas where the basement membrane rests as the only barrier between the filtrate and the peritubular interstitium. This permits for back leak of the filtrate, particularly under circumstances where the pressure in the tubule is increased due to intratubular obstruction resulting from cellular debris in the lumen interacting with proteins such as fibronectin which enter the lumen (Zuk et al, 2001). Results of this work indicated the presence of necrotic and apoptotic cell death, necrotic pathways may be due to decline in cellular ATP concentrations which lead to diminish of Na+-K+ ATPase activity and net Na+ influx accompanied with swelling of the cytosolic compartment. The accompanied swelling of cells is believed to be precursor to necrotic death (Basile et al, 2012).

The interstitial nephritis that was observed in paracetamol treated rats of this study may be attributed to the continuing glomerular damage that may generates capillary hypertension, with enhanced glomerular filtration and elapse of proteins into the tubular fluid. The glomerular proteinuria may also elevates the formation of angiotensin II and stimulates discharge of inflammatory mediators (cytokines and chemokines), that trigger the renal interstitial build-up of mononuclear cells.

The initial neutrophil recruiting is probably substituted by macrophages and T lymphocytes, which release the immune response, forming interstitial nephritis (Vianna et al, 2011) or, this may be attributed to reduction in cyclooxygenase by paracetamol, causing shunting of arachidonic acid precursor into lipoxygenase pathway, promoting generation of inflammation stimulating metabolites of eicosapentaenoic acid which functions as lymphokinase, resulting in recruitment of T-lymphocytes and propagation of the inflammatory process (Yasmeen et al, 2007). Glomeruli hypercellularity may


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be due to the proliferative glomerulornephritis which may be resulted from proliferation of endothelial cells, mesangial cells or infiltration with inflammatory cells (Kumar et al, 2012).

Regarding our findings for rats that were treated with green tea extract (Group III), at which glomerular and tubular lesions were described like those in rats treated with paracetamol (Group II), similar results of Isbrucker and his team (2006) are consistent with our observations as they revealed that high dose of EGCG stimulated severe proximal tubular necrosis in male beagle dogs and degeneration of some tubules in the females.

Administration of paracetamol followed by green tea extract in this study (Group IV) induced extensive alterations of glomeruli and tubular structures for renal tissues. This may due to the presence of renal corpuscles appeared without glomeruli and degeneration of glomeruli in some renal corpuscles and the existence of protein casts, necrosis and apoptosis in cells of proximal and distal tubules as well as squamation of cells of renal tubules together with the presence of cystic spaces.

PAS staining for glycogen content: Histochemical changes:

Nonsteroidal anti-inflammatory drugs (NSAIDs) are common causes of the secondary

membranous glomerulonephritis (Ponticelli and Glassock, 2014). The membranous glomerulonephritis is characterized by the thickening of glomerular basement membrane (GBM); this thickening is owed to incidence of subepithelial accumulations and extra matrix material laid down by damaged podocytes (Kwatra and Prasher, 2013).

The current study showed increase in glycogen content in renal tissue of rats treated with 400mg/ 200g paracetamol (Group II) especially in the glomerular basement membrane (GBM) which may refer to the occurrence of secondary membranous glomerulonephritis. These results disagree with Refaat and Mady (2008), who found weak PAS reaction in renal tissue after treatment with a single intraperitoneal dose of paracetamol. This disagreement may be due to dose administration of their study. On the other hand, same results were observed as well in rats treated with green tea extract and paracetamol followed by green tea extract (Group IV).

In spite of paracetamol (acetaminophen) is generally not considered as an NSAID because it has only little anti-inflammatory activity (Hinz et al, 2008), it treats pain mainly by blocking COX-2 (cyclooxygenase-2) mostly in the central nervous system, but not much in the rest of the body. Most NSAIDs inhibit the activity of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), and thereby the synthesis of prostaglandins and thromboxanes. It is thought that inhibiting COX-2 leads to the anti-inflammatory, analgesic and antipyretic effects (Clive et al, 1998). It seems that paracetamol may causing the same histopathological lesions due to its ability to inhibit synthesis of prostaglandins and thromboxanes and this may explicate our findings regarding the secondary membranous glomerulonephritis.

Masson's trichrome stain:

In this investigation, the renal tissue after treatment with paracetamol, green tea extract and

paracetamol followed by green tea extract exhibited glomerulosclerosis and tubulo-interstitial fibrosis. The basic cellular processes resulting in these pathological alterations are more complex. In most cases of tissue injury, damaged cells are substituted by cells of the same kind and/or fibrous tissue after the resolution of the inflammatory response. (Masson & Verhoff, 2009)

Kidney has a native ability for self-healing following ischemic or toxic insults which induce cell death and can possibly recover completely after enduring acute damage (Lawson et al, 2015). This process of renal tissue repair undergoes a cascade of events to heal the injury. These processes involve kidney resident cell activation, which stimulates the production and discharging of pro-inflammatory cytokines. The gradients of chemotactic cytokines afford a directional signal for regulating the infiltration of inflammatory monocytes/macrophages and T cells to the damaged positions. Depending on the etiology of renal injury, glomerular or interstitial infiltrated inflammatory cells become stimulated, and generate deleterious particles such as reactive oxygen species (ROS), in addition to fibrogenic and inflammatory cytokines (Liu, 2006). These, sequentially, trigger mesangial


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cells, fibroblasts, and tubular epithelial cells to undergo phenotypic stimulation or alteration and generate a great amount of extra cellular matrix (ECM) constituents.

Normal and new forms of ECM proteins are then accumulated in the extracellular compartment, and they are regularly cross-linked and become withstanding to degradation. Incessant accumulation of ECM produces fibrous scars and alters the fine construction of renal tissues, causing the collapse of renal parenchyma and the missing of kidney function (Liu, 2006). Immunohistochemical studies:

In the current study, administration of paracetamol induced apoptosis in renal tubular cells and

glomeruli which was demonstrated by increasing the expression of active caspase-3. In accordance with our results, Ahmad et al, (2012), as well as Hassan and A. Rahman (2016) observed high positive expression of caspase-3 in renal tissue following treatment with a high dose of paracetamol. Additionally, this result was reinforced with a previous study that indicates the development of caspase-3 cleavage product in Western blots and the development of caspase-3–like activity in cell extracts treated with paracetamol (Lorz et al, 2004).

Identically, rats treated with green tea extract exhibited strong positive expression of active caspase-3 in renal tissue. This finding conflicts with many previous investigations. Rehman et al, (2013) observed that cleaved caspase-3 was scarcely detectable after administration of green tea polyphenols. Besides, Abozalam et al, (2016) showed that treatment with green tea extract alone caused no immuno-reactivity of caspase-3. The same result was obtained in renal tissue of rats treated with paracetamol followed by green tea extract.

Paracetamol stimulates caspase-dependent apoptosis of tubular cells devoid of characteristic mitochondrial dysfunction or contribution of Bax (lorz et al, 2004). Paracetamol nephrotoxicity is an example of participation of the endoplasmic reticulum (ER) in apoptosis. ER-initiated apoptosis is induced by disorders in calcium homeostasis or depositions of misfolded proteins and various signaling pathways developing to promote cell death via caspase-dependent and independent ways, comprising the recruitment of the mitochondrial pathway (Ferri and Kroemer, 2001; Xu et al, 2005 & Breckenridge et al, 2003). Molecular responses distinctive of participation of the ER in apoptosis involve the expression of CHOP/GADD153, a transcription factor that reduce Bcl-2 levels, and activation of ER-associated caspase-12 (Nakagawa et al, 2000 & McCullough et al, 2001). After its stimulation at the ER, caspase-12 may directly process downstream caspases in the cytosol or target other substrates that impact the evolution of apoptosis (Breckenridge et al, 2003). It was noted that caspase-12 can directly cleave procaspase-9 which cause caspase-9-dependent activation of caspase-3 (Morishima et al., 2002 & Rao et al., 2002).

In conclusion, the histopathological and immunohistochemical investigation of renal tissues confirmed the biochemical assessment results which explicate the incidence of severe renal damage in rats treated with paracetamol, green tea extract and paracetamol followed by green tea extract in addition to rats that were left to recover. We can strongly recommend that administration of both paracetamol and green tea extracts should be prescribed under medical restrictions and using paracetamol followed by green tea extract should be avoided due to their injurious obstacles on the structural details and hence the functional excretory and secretory mechanisms of kidney.


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Fig. 4: A section in the cortex of kidney of control rat (Group I), showing normal architecture of glomerular tuft (g), Bowman's space ( ), Bowman's capsule (thick arrow), proximal tubule (pt) and distal tubule (dt) (H&E., × 400)

Fig. 5: A section in the medulla of kidney of control rat (Group I), showing normal architecture of medullary tubules with the descending limb of loop of Henel (dl ), ascending limb of loop of Henel (al) and collecting tubules (ct). (H&E., × 400)

Fig. 6: A section in the cortex of kidney of paracetamol treated rat (Group II), showing a glomerular ischemic collapse (thick arrow) (H&E., × 400)

Fig. 7: A section in the cortex of kidney of paracetamol treated rat (Group II), showing degeneration of the glomeruli in some renal corpuscles (thick arrows) (H&E., × 400)

Fig. 8: A section in the cortex of kidney of paracetamol treated rats (Group II), showing an enlargement of renal corpuscle (thick arrow) with the widening in Bowman's spase (*) (H&E., × 400)

Fig. 9: A section in the cortex of kidney of paracetamol treated rats (Group II), showing multifocal tubulo-nephritis with marked lymphocytic infiltration (thick arrows) (H&E., × 400)

pt

dt

g

dl

al

ct

*


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Fig. 10: A section in the cortex of kidney of paracetamol treated rat (Group II), showing multifocal tubulo-nephritis with marked lymphocytic infiltration (thick arrows) and vaculated cytoplasm with necrotic nuclei (thin arrows) (H&E., × 400)

Fig. 11: A section in the cortex of kidney of paracetamol treated rat (Group II), showing protein casts (thick arrows) and squamation of the tubular cells (thin arrows) (H&E., × 400)

Fig. 12: A section in the cortex of kidney of paracetamol treated rat (Group II), showing necrotic cells (thick arrows) and cystic spase (*)(H&E., × 400).

Fig. 13: A section in the cortex of kidney of paracetamol treated rat (Group II), showing apoptic cells(thick arrows) and intertubular congestion (thin arrows)(H&E., × 400).

Fig. 14: A section in the cortex of kidney of green tea extract treated rat (Group III), showing squamation of the tubular cells( thin arrows) (H&E., × 400)

Fig. 15: A section in the cortex of kidney of paracetamol followed by green tea extract treated rat (Group IV), showing vaculated cells with necrotic nuclei (thin arrows) (H&E., × 400)

*


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Fig. 20: A section in the cortex of kidney of control rat (Group I), showing normal amount of collagen fibers (Blue) around renal tubules, Bowmann's capsule (thin arrows) and in the glomerulus (thick arrow) (Masson's trichrome., × 400)

Fig. 21: A section in the cortex of kidney of paracetamol treated rat (Group II), showing an increasing amount of collagen fibers around renal tubules (thin arrows) and in the glomerulus (thick arrow) (Masson's trichrome., × 400)

Fig. 16: A section in the cortex of kidney of control rat (Group I), showing normal accumulation of carbohydrates in the renal tubules (thin arrow) and the glomerulus (thick arrow) ( PAS.,× 400)

Fig. 17: A section in the cortex of kidney of paracetamol treated rat (Group II), showing increasing accumulation of carbohydrates in renal tubules (thin arrows) ( PAS.,× 400)

Fig. 18: A section in the cortex of kidney of green tea extract treated rat (Group III), showing increasing accumulation of carbohydrates in the intertubular tissues (thin arrows) ( PAS.,× 400)

Fig. 19: A section in the cortex of kidney of paracetamol recovery rat (Group V), showing increasing accumulation of carbohydrates in glomerulus (thick arrows) and tubular cells as well (thin arrows) ( PAS.,× 400)


883

Fig. 22: A section in the cortex of kidney of paracetamol treated rat (Group II), showing highly increased amount of collagen fibers between renal tubules (thin arrows) (Masson's trichrome., × 400)

Fig. 23: A section in the cortex of kidney of green tea extract treated rat (Group III), showing massive increasing in the collagen fibers in basement membranes of renal tubules (thin arrows) and glomerulus (thick arrows) (Masson's trichrome., × 400)

Fig. 24: A section in the cortex of kidney of green tea extract treated rat (Group III), showing obvious increase in the collagen fibers among renal tubules (thin arrows) (Masson's trichrome., × 400)

Fig. 25: A section in the cortex of kidney of paracetamol followed by green tea extract recovery rat (Group VI), showing increase in the collagen fibers in glomerulus (thin arrows) and basement membranes of renal tubules (thick arrow) (Masson's trichrome., × 400)

Fig. 26: A section in the cortex of kidney of control rat (Group I), showing negative reaction for caspase-3 in the glomerulus (thick arrow) and renal tubules (thin arrows) (Immunolabeling for caspase 3., × 400)

Fig. 27: A section in the cortex of kidney of paracetamol treated rat (Group II), showing high caspase-3 experssion in the renal tubules (thin arrows) (Immunolabeling for caspase 3., × 400)


884

Fig. 28: A section in the cortex of kidney of green tea extract treated rat (Group III), showing moderate caspase 3 immunolabeling in the glomeruli (thin arrows) (Immunolabeling for caspase 3., × 400)

Fig. 29: A section in the cortex of kidney of paracetamol followed by green tea extract treated rat (Group IV), showing obvious higher positive reaction for caspase-3 in the glomerulus (thick arrow) and renal tubules (thin arrows) (Immunolabeling for caspase 3., × 400)

Fig. 30: A section in the cortex of kidney of paracetamol recovery rat (Group V), showing a mild positive reaction for caspase-3 in the glomerulus (thin arrow) and renal tubules (thick arrows) (Immunolabeling for caspase 3., × 400)

Fig. 31: A section in the cortex of kidney of paracetamol followed by green tea extract recovery rat (Group VI), showing strong positive reaction for caspase-3 in the glomerulus (thin arrow) and renal tubules (thick arrows) (Immunolabeling for caspase 3., × 400).

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