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COX-1 AND COX-2 EXPRESSION IN KIDNEYS FROM CAPSAICIN TREATED RATS. 389

Introduction

Capsicum annuum L. is an important plant frequently used by humans, in the pharmaceutical industry, chemistry, medicine, and veterinary medicine. Hot green and red peppers are commonly used spices in human food. Capsaicin is the majör active ingredient of hot green and red peppers of the plant genus Capsicum [8].

Some available studies state that capsaicin (CAP) decreases the triglyceride concentrations in liver and serum and the amount of fatty tissues by increasing lipid peroxidation and also that capsaicin has an inhibitor effect on the glycogen metabolism of skeletal muscles in an in vitro environment [10, 11]. Capsaicin also decreases the risk of developing arteriosclerosis by decreasing cholesterolemia, and serum triglyceride concentrations [10, 19].

Other studies aim to clarify the direct relation between

capsaicin and pain sensation [2, 3, 23]. Capsaicin is included in the group of analgesic substances and is efficient on primary sensory neurons [2, 3, 27]. It is also specified that it is also used in post hepatic neuropathy, diabetic and other neuropathic pains by eliminating the sensitivity of such neurons [2, 3, 27].

A German chemist, Felix Hoffman, had produced acetylsalicylic acid (aspirin), which is the first non-steroid anti-inflammatory drug (NSAID) [25], for the first time in 1893. Although the NSAIDs have been considerably used in the last century, they are the drugs for which the mechanisms of action were not completely known until the structure of COX enzyme was identified in 1971. Until 1990, only one type of cyclooxygenase was considered but because of the considerable extend of molecular biology techniques, 2 types of cyclooxygenases, constitutive (also called COX-1) and inducible (also called COX-2) types, were identified.

SUMMARY

The objective of this study was to investigate the effects of capsaicin on the renal distribution of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in pubere rats. The expression of the 2 enzymes was analyzed using immunohistochemistry in 50 days old rats daily treated with capsaicin (1 mg/kg, subcutaneously) diluted in 10% ethanol, 1% Tween 20 and 80% distilled water for one week, as well as in sham rats (only injected with the vehicle) and in not injected control rats (n = 10 in each group). Weight growth was significantly deleted in the treated rats but no kidney damage was evidenced by conventional histology. The COX-1 and COX-2 localisations were similar in all rats but the COX-2 expression intensity was markedly depressed in the proximal and distal tubules, macula densa and loops of Henle from the capsaicin treated rats whereas the COX-1 expression appeared increased in proximal tubules compared to the sham and control rodents. These results demonstrate that capsaicin which is already known to affect gastro-intestinal, cardiovascular and respiratory systems, also interfere with the expression of cyclooxygenases in kidney in rats.

Keywords: rats, capsaicin, kidney, cyclooxygenases, immunohistochemistry.

RESUME

Distribution immunohistochimique de COX-1 et COX-2 dans le tissu rénal de rats pubères traités par la capsaïcine

L’objectif de cette étude a été d’évaluer les effets de la capsaïcine sur l’expression des cyclooxygénases (COX), COX-1 et COX-2, dans le parenchyme rénal de rats pubères. L’expression de ces 2 enzymes a été analysée par immunohistochimie chez des rats de 50 jours traités quotidiennement par la capsaïcine (1 mg/kg par voie sous-cutanée) diluée dans 10% d’éthanol, 1% de tween 20 et 80% d’eau distillée pendant une semaine, ainsi que chez 2 groupes de rats témoins, le premier dans lequel les animaux ont été injectés uniquement par le diluent, le second dans lequel les rats n’ont reçu aucun traitement (chaque groupe était composé de 10 animaux). La croissance pondérale a été significativement ralentie chez les rats traités mais aucune lésion rénale n’a été révélée par histologie. La localisation des enzymes COX-1 et COX-2 a été identique chez tous les rats mais l’intensité de l’expression de COX-2 a été bien plus faible dans les tubules proximaux et distaux, la macula densa et les anses de Henlé chez les animaux traités alors que celle de COX-1 est apparue augmentée dans les tubules proximaux par rapport aux animaux témoins. Ces résultats montrent que la capsaïcine, déjà connue pour affecter les systèmes gastro-intestinaux, cardiovasculaires et respiratoires, interfère également avec l’expression des cyclooxygénases rénales chez le rat.

Mots-clés : Rats, capsaïcine, rein, cyclooxygénases, immunohistochimie.

Immunohistochemical distribution of COX-1 and COX-2 in the renal tissue of pubere rats treated with capsaicinS. ELIS YILDIZ1*, G. NUR2, M. NAZLI3, M. SOZMEN4

1School of Health Sciences, Kafkas University, Pasacayır Campus, 36100 Kars, TURKEY2Department of Biology, Faculty of Arts and Sciences, Kafkas University, Pasacayır Campus, 36100 Kars, TURKEY3Histology and Embriyology, Faculty of Veterinary Medicine, Mehmet Akif Ersoy University, Istiklal Campus, 15030 Burdur, TURKEY4Department of Pathology, Faculty of Veterinary Medicine, Ondokuz Mayıs University, Kurupelit Campus, 55200 Samsun, TURKEY

Corresponding author : sevdaelis36@hotmail.com

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Although COX-1 and COX-2 catalyze the same reactions, they have different structures and functions [5]. Besides, a third form called as COX-3, was recently found [26]. COX-1 has a protective effect mostly in the tissues and is efficient in regulating physiological protective functions such as the platelet aggregation and homeostasis in gastrointestinal system and kidneys and the COX-2 enzyme has the exact opposite effect [7]. While COX-1 is expressed in the kidney and especially in the veins, smooth muscles and collecting drain cells, COX-2 is expressed in macula densa, cortical thick  ascending limb cells of the loop of Henle and the medullary interstitial cells [1, 14].

The purposes of this study were to determine the immunohistochemical localisation of Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) in rat renal tissues by immunohistochemistry and to identify possible difference in COX-1 and COX-2 expression in pubere rats as example for a development period.

Material and Methods

ANIMALS AND EXPERIMENTAL DESIGN

This study, approved by the Animal Testing Local Ethics Committee of Kafkas University, was conducted on 30 male pubere (50 days old) Sprague-Dawley rats. Animals were kept in standard cages in an environment at 22 ± 2°C, and were exposed to 12-hour sunlight, and 12-hour darkness and were weighed every day for a week before they were treated.

The rats were randomly divided into three groups, experimental, sham and control groups. Capsaicin (CAP) (Sigma M 2028) was prepared daily by dissolution in 10% ethanol, 1% Tween 20 (Merck M8170772100) and 80% distilled water and was subcutaneously injected in rats from the experimental group at the same time every day for a week using an insulin injector at 1 mg/kg of body weight according to previous studies of MORAN et al. [15] and TÜTÜNCÜ et al. [24]. Rats from the sham group received only the diluent (10% ethanol, 1% Tween 20 and 80% distilled water) according to the same modalities than in the experimental group whereas controls had no application. At the end of the week treatment, all rats were weighed, and then killed using deep ether anaesthesia before removing kidneys. Weight gains (expressed in %) were calculated using the formula: 100 x (Wi – W0)/W0 where Wi was the weight determined at the 7th or at the 14th days and W0 the initial weight.

KIDNEY ANALYSES

After removal, kidney tissues were immediately fixed in a 10% formaldehyde solution, were routinely embedded in paraffin blocks and 5 μm thick serial sections were performed for conventional histology. The 5 μm thick sections were stained with Crossman’s triple stain for histological investigations and other sections were immunostained for evidencing COX-1 and COX-2 in kidney [13].

For immunohistochemistry, the sections were incubated in 3% H202 for 15 minutes to inhibit endogenous peroxidase activity after undergoing deparaffinization and rehydration. Maximum heat was applied in a microwave for 10 minutes in citrate buffer solution to reveal antigens, after being washed with PBS (Phosphate Buffer Saline). Blocking solution A (Invitrogen Histostain plus Broad Spectrum (AEC) Ref. 85.9943) was dripped onto the sections to reduce nonspecific binding of antibodies. Sections were then incubated with primary antibodies (anti-COX-1 (5F6/F4, ab695, abcam) and anti-COX-2 (M-19, sc-1747, Santa Cruz)) diluted to 1:200 in PBS at room temperature for one hour. Only PBS was dripped on the tissues of the negative control group. Streptavidin-biotin peroxidase technique was used after primary antibody incubation [9]. For this purpose, a Broad Spectrum Antibody (Invitrogen Histostain plus Broad Spectrum (AEC) Ref. 85.9943) antibody, counter to the type produced by the primary antibody, was added onto sections, and left at room temperature for 15 minutes. Streptavidin-HRP (Horse Radish Peroxidase) (Invitrogen Histostain plus Broad Spectrum (AEC) Ref. 85.9943) was dripped on the sections, and then incubated at room temperature for 15 minutes. After adding 3-Amino-9-Ethylcarbazole (AEC) Staining Kit solution, the sections were placed under a light microscope to check for immunoreactivity; the reaction was inactivated with distilled water in accordance with the immunoreactivity status. It was then dipped in haematoxylin for negative staining. A lamella was covered the kidney tissue after dripping water-based glue on them (Labvision, Large Volume Vision Mount, TA-060-UG). The slides were examined using light microscope, and their photographs were taken. Scoring was conducted using a semi-quantitative method on an area of the sections, based on criteria of the percentage of stained cells and the degree of staining. The degree of staining was evaluated as follows; 0 (no staining), 1+ (weak), 2+ (moderate) and 3+ (strong staining).

STATISTICAL ANALYSIS

Data were expressed as mean ± standard deviation and were compared using one-way ANOVA and Tukey HSD test. Differences were considered as significant when p value was less than 0.05.

Results

As shown in Table I, it was observed that the weekly weight gains were similar in the 3 groups (experimental, sham and control) before treatment. By contrast, at the end of the week treatment, weight gains have considerably differed according to the groups: weights measured on day 14 (157,78 ± 26,65 g) in rats treated with capsaicin (CAP) were slightly lower than those determined on day 7 (158,02 ± 24,65 g) and the overall weight gain was only 9.05% whereas it reach 17.46% and 25.60% in the sham and control groups, respectively (experimental group vs. sham and control groups: p < 0.05).

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COX-1 AND COX-2 EXPRESSION IN KIDNEYS FROM CAPSAICIN TREATED RATS. 391

Histologically, normal kidney structures were observed in all rats (experimental, sham and control groups). No difference was obtained between the 3 groups.

COX-1 and COX-2 immunoreactivities were observed by immunohistochemistry in kidneys from all rats (Table II). A weak (1+) COX-1 expression was seen in the medulla in all groups and in the proximal tubules of the sham and the control groups (figure 1B), whereas it was moderate (2+) in those of the capsaicin treated rats (figure 1A). No COX-1 immunoreactivity was detected in the glomeruli, vascular endothelium and distal tubules in the 3 groups. On the other hand, COX-2 immunolabelling was moderate to dense (proximal and distal tubules) and dense (macula densa and loops of Henle) in the renal structures from sham and control rats (figures 2C and 2D) and remained moderate in the experimental group except in some proximal convoluted tubules (figures 2A and 2B).

DiscussionIn the present study, it was observed that capsaicin

treatment for one week has significantly delayed weight growth in pubere rats. TUTUNCU et al. [24] also reported that the body weight of rats receiving capsaicin was lower in comparison to the control rats and numerous studies support the view that capsaicin reduces body weight gains [4, 11, 15, 17, 18, 21]. The effects on weight growth may be related to

the increases in carbohydrate and lipid mobilisation and catabolism and in liver enzyme activities [24].

Normal kidney histological results were obtained in rats treated or not with capsaicin in the current study, suggesting that capsaicin is probably not nephrotoxic.

It was stated that capsaicin or chili pepper administration to rats for a long time is effective in the treatment of the lesions produced by agents such as alcohol, aspirin or stress in the stomach, and plays a protective and analgesic role against damages [22]; it was determined that afferent neurons that are sensitive to the capsaicin are effective on this situation [22]. Cytokines, growth factors, tumour necrosis agents, pro-inflammatory stimulants such as bacterial endotoxin rapidly causes COX-2 expression in inflammatory cells [20]. Prostaglandins (PGs) produced by COX-2 play a major role in inflammatory reactions and are responsible for characteristic inflammatory symptoms such as rubor, fever, tubercle, pain and functional loss [20]. As capsaicin acts on primary sensory neurons, the analgesic effect of capsaicin may be mediated by removing sensitivity of these neurons [2, 3, 27].

In humans, mice and rats, COX-1 immunoreactivity is evidenced in proximal convoluted tubules both in cortex and medulla but the enzyme is expressed neither in glomerulus nor in epithelial cells of distal tubules [1, 14, 28]. In this

Experimental group Sham group Control group

Initial weight (day 0) (g)Weight on day 7 (g)

Weight gain on day 7 (%)Weight on day 14 (g)

Weight gain on day 14 (%)

144.68 ± 23.65158.02 ± 24.65

9.26 ± 1.38158.02 ± 24.65b

9.05 ± 1.39b

140.42 ± 31.70156.13 ± 29.23

11.10 ± 2.06156.13 ± 29.20b

17.46 ± 3.25a

159.27 ± 22.69181.03 ± 25.21

13.69 ± 1.89200.10 ± 26.45a

25.60 ± 3.32a

Weight gains were calculated using the formula: 100 x (Wi – W0)/W0 where Wi was the weight determined at the 7th or at the 14th days and W0 the initial weight.

Different superscripts a,b in the same raw indicate significant difference (p < 0.05 or more) between groups.

Table I: Weight variations in pubere rats subcutaneously treated daily at the 7th day for one week with capsaicin (1mg/kg in 10% ethanol, 1% Tween 20 and 80% distilled water, experimental group), with diluent (10% ethanol, 1% Tween 20 and 80% distilled water, sham group) or no treated (control group) (10 rats per group). Results are expressed as means ± standard deviations.

Renal structuresCOX-1 COX-2

Experimental group Sham and Control groups Experimental group Sham and Control

groups

GlomerulusProximal tubulus

Distal tubulusMacula densa

Loop of Henle

NoModerate

NoNo

Weak

NoWeak

NoNo

Weak

NoModerate / Dense

ModerateModerateModerate

NoModerate / Dense

ModerateDenseDense

Table II: Semi-quantitative COX-1 and COX-2 expression detected by immunohistochemistry in kidneys from pubere rats subcutaneously treated daily at the 7th day for one week with capsaicin (1mg/kg in 10% ethanol, 1% Tween 20 and 80% distilled water, experimental group), with diluent (10% ethanol, 1% Tween 20 and 80% distilled water, sham group) or no treated (control group) (10 rats per group).

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Figure 1: Kidney section from a rat treated with capsaicin (1 mg/kg/day SC for 7 days). Moderate COX-1 immunoreactivity is seen in the proximal convoluted tubules (A.); Kidney section from a control rat (no treatment). Weak COX-1 immunoreactivity is seen in the proximal convoluted tubules (B.). G: Glome-rulus; PCT: Proximal convoluted tubule; TD: Distal tubule.

Figure 2: Kidney section from a rat treated with capsaicin (1 mg/kg/day SC for 7 days). The COX-2 expression was dense in some proximal convoluted tubules and moderate in distal tubules and macula densa (A.) and moderate in the loops of Henle (B.); Kidney section from a control rat (no treatment). The COX-2 immunoreactivity was moderate in distal tubules, moderate to dense in proximal tubules and dense in macula densa (C.) as well as in the loops of Henle (D.). G: Glomerulus; PCT: Proximal convoluted tubule; TD: Distal tubule; MD: Macula densa; LH: Loop of Henle. Immunohistochemistry, Bar:100 µm.

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study, the presence of COX-1 was similar. The presence of low amount of COX-2 mRNA was determined in the renal tissues in 1993 for the first time [16]. In situ hybridisation and immunohistochemical methods demonstrated that the COX-2 was located in the macula densa cells, in distal tubules, medullary interstitial cells, and in the epithelial cells of the thick ascending limb of the loop of Henle in the cortex [6]. In humans, the COX-2 protein has been revealed in the endothelium and smooth muscle cells in arteries and veins and in podocytes in glomeruli, as well as in the macula densa cells [12].

The COX-2 expression in the present study was also found in all parts of the nephron except in glomerulus. However, in the present study, the COX-1 expression in rats treated with capsaicin for one week was strengthened in the proximal tubules and the COX-2 expression was depressed mainly in macula densa, distal tubules and loops of Henle compared to the rodents not receiving the analgesic substance (sham and control groups), indicating that the inflammatory prostaglandin synthesis involving the COX-2 enzyme was markedly alleviated in kidneys.

As a conclusion, it is well known that Non-Steroid Anti-Inflammatory Drugs have very different activities in kidneys. Studies conducted with specific COX-2 inhibitors such as capsaicin, would open up the route to clarify with more accuracy the particular roles of COX-1 and COX-2 in renal physiology.

Notes

This study has already been presented at the XIth National Histology & Embryology Congress (With International Contribution), 16-19 May 2012, pp.:34 (Abstr.) Denizli Turkey.

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