ABSTRACT

Aim:

Cisplatin is one of the effective antineoplastic drugs widely used in the treatment of many types of cancer. Cisplatin has harmful effects such as nephrotoxicity, ototoxicity and cardiomyopathy. Quercetin is an antioxidant of the flavonoid group. In this study, it was aimed to investigate the therapeutic effects of quercetin against cisplatin-induced kidney damage in rats.

Materials and Methods:

Twenty-eight male Wistar albino rats were randomly selected and divided into 4 groups: Group 1: Control (no application), Group 2: Quercetin (25 mg/kg/7 days/intraperitoneal), Group 3: Cisplatin (7 mg/kg/single dose/ intraperitoneal), Group 4: Cisplatin+quercetin (7 mg) /kg/single dose/ intraperitoneal cisplatin followed by 25 mg/kg/7 days/ intraperitoneal quercetin). After routine histological follow-up, hematoxylin eosin and periodic acid-schiff staining were performed. Histopathological damage score was calculated. Caspase-3 immunostaining was performed and scored.

Results:

Control and quercetin groups had normal histological appearance. In the cisplatin group, dilatation of the tubules, epithelial shedding, vacuolization of the tubular epithelial cells, and loss of microvilli in the proximal tubules were detected. In addition, infiltration areas were also found in places. In addition, an increase in caspase-3 immunostaining intensity was detected in this group (p=0.000). Histopathological findings were significantly reduced in the cisplatin+quercetin group compared to the cisplatin group (p=0.001).

Conclusion:

In this study, we think that quercetin is histopathologically beneficial in the treatment of cisplatin-induced kidney damage.

INTRODUCTION

Cisplatin is one of the potential and widely used drugs in the treatment of various solid cancers such as testicular, ovarian, head and neck, bladder, lung, lymphoma, cervical cancer, and melanoma¹. The anticarcinogenic effect of cisplatin occurs through the interaction with purine bases on DNA, causing deoxyribo nucleic acid (DNA) damage and activation of signal transduction pathways that lead to apoptosis (programmed cell death)¹. It has been shown that cisplatin plays a role in the formation of reactive oxygen species, thus inducing apoptosis via intrinsic caspases and causing mitochondrial dysfunction². Cisplatin fights tumors through the induction of apoptosis mediated by activation of various signal transduction pathways, including calcium signaling, death receptor signaling, and activation of mitochondrial pathways². The most important limiting factor in the use of anticarcinogenic drugs is their side effects. Cisplatin is characterized by toxic effects such as nephrotoxicity, cardiotoxicity, hepatotoxicity, neurotoxicity and myelosuppression³.

Quercetin (3,3’,4’,5,7-pentahydroxyflavone), a member of the flavonoid family, is one of the polyphenolic compounds found in various foods⁴. Quercetin is found in onions, apples, strawberries, cauliflower, cabbage and many other foods⁵. Studies have shown that quercetin has anticarcinogenic effects, but it reduces oxidative damage by inhibiting the activity of xanthine oxidase, a form of xanthine oxidoreductase, which is a type of enzyme that produces reactive oxygen species, and has anti-inflammatory activity by inhibiting the production of tumor necrosis factor alpha depending on the dose^6-8.

Caspase-3, a cysteine-aspartic acid protease, is not activated until it is cleaved by initiator caspases during the apoptotic flux⁹. After activation, it cuts non-caspase target proteins in cells from their specific regions^10,11. Thus, it plays an important role in programmed cell death¹².

In this study, it was aimed to histopathologically examine the therapeutic effects of quercetin against cisplatin-induced kidney damage in rats.

GİRİŞ

Sisplatin testis, over, baş ve boyun, mesane, akciğer, lenfoma, serviks kanseri, melanom gibi çeşitli solid kanserlerin tedavisinde potansiyel ve yaygın olarak kullanılan ilaçlardan biridir¹. Sisplatinin antikanserojen etkisi, deoksiribo nükleik asit (DNA) üzerindeki pürin bazları ile etkileşime girerek DNA hasarını oluşturması ve apoptoza (programlanmış hücre ölümü) yol açan sinyal iletim yollarının aktivasyonu üzerinden gerçekleşir¹. Sisplatinin reaktif oksijen türlerinin oluşumunda rol oynadığı böylece intrinsik kaspazlar yoluyla apoptozisi indüklediği ve mitokondriyal disfonksiyona neden olduğu gösterilmiştir². Sisplatin, kalsiyum sinyali, ölüm reseptörü sinyali ve mitokondriyal yolların aktivasyonu dahil olmak üzere çeşitli sinyal iletim yollarının aktivasyonunun aracılık ettiği apoptoz indüksiyonu yoluyla tümörlerle savaşmaktadır². Antikanserojen ilaçların kullanımında sınırlayıcı en önemli faktör yan etkileridir. Sisplatin nefrotoksisite, kardiyotoksisite, hepatotoksisite, nörotoksisite ve miyelosupresyon gibi toksik etkilerle karakterizedir³.

Kuersetin (3,3’,4’,5,7-pentahidroksiflavon), flavonoid ailesinin bir üyesi olmakla birlikte çeşitli yiyeceklerde bulunan polifenolik bileşiklerden biridir⁴. Kuersetin soğan, elma, çilek, karnabahar, lahana ve diğer birçok gıdada bulunur⁵. Yapılan çalışmalar, kuersetinin antikanserojenik etkilere sahip olduğunu göstermekle birlikte, reaktif oksijen türleri üreten bir tür enzim olan ksantin oksidoredüktazın bir formu olan ksantin oksidaz aktivitesini inhibe ederek oksidatif hasarı azalttığı ve proinflamatuvar sitokinlerden biri olan tümör nekroz faktörü alfanın üretimini doza bağımlı bir şekilde inhibe ederek antiinflamatuvar aktiviteye sahip olduğunu göstermektedir^6-8.

Bir sistein-aspartik asit proteazı olan caspase-3, apoptotik akış sırasında başlatıcı kaspazlar tarafından parçalanana kadar aktive olmaz⁹. Aktive olduktan sonra hücrelerdeki kaspaz ailesinden olmayan hedef proteinlerini spesifik bölgelerinden kesmektedir^10,11. Böylece, programlanmış hücre ölümünde önemli bir rol üstlenmektedir¹².

Bu çalışmada, sıçanlarda sisplatin ile oluşturulan böbrek hasarına karşı kuersetinin tedavi edici etkilerinin histolopatolojik olarak incelenmesi amaçlanmıştır.

MATERIALS AND METHODS

Groups

Approval for the study was obtained from the Animal Experiments Local Ethics Committee of İnönü University Medical Faculty (ethics committee no: 2012/A-103, date: 09.06.2012). Animal rights were protected in line with the principles of the ‘Guide for the Care and Use of Laboratory Animals’. In line with these principles, 28 healthy male Wistar albino rats, 28-30 days old and weighing 300-350 grams, obtained from Inonu University Faculty of Medicine Experimental Research Laboratory, were used. The rats were left in a room with 12 hours of light and 12 hours of darkness, in a daylight rhythm, in a ventilated environment with a temperature of 21 ºC and a humidity of 55-60% for 21 days. They were fed ad libitum with standard pellet feed and tap water in special cages. Randomly selected rats were divided into 4 equal groups, each with7 animals. Groups were organized as Group 1: Control (no application), Group 2: Quercetin (25 mg/kg/intraperitoneal for 7 days), Group 3: Cisplatin (single dose 7 mg/kg/intraperitoneal), Group 4: Cisplatin+quercetin (single dose 7 mg/kg/intraperitoneal cisplatin, then 25 mg/kg/intraperitoneal quercetin for 7 days). Quercetin (CAS Number: 117-39-5) and cisplatin (CAS number: 15663-27-1) were obtained from Sigma Chemical Co. (St. Louis, MO). Cisplatin and quercetin doses used in this study were determined according to previous studies in the literature^13,14.

Histopathological Analysis

At the end of the experiment, the rats were sacrificed under ketamine (90 mg/kg/intraperitoneal) anesthesia. Their kidneys were removed, rinsed with saline and fixed in 10% neutral buffered formalin solution for histological evaluation. After the tissues were fixed in formalin for 72 hours, they were dehydrated by passing through increasing alcohol series (70%, 80%, 96% and 100%). Finally, they were kept in xylene and embedded in paraffin. Tissue sections with 5 μm thickness were obtained using a fully automated microtome. Hematoxylin-eosin staining method was used to examine the general histological structure and periodic acid-schiff (PAS) staining method was used to observe glycogen accumulation.

Histopathological Evaluation

Evaluation was done in a double-blind fashion by a histologist in the study. Renal damage was determined semi-quantitatively according to the degree and extent of histopathological changes. Tissues were examined for dilation of tubules, shedding of tubular epithelium, vacuolization of tubular epithelial cells, peritubular infiltration and loss of microvillus in proximal tubules. All sections were examined at 20X magnification in 10 different fields and scored as 0 (no change), 1 (mild), 2 (moderate), and 3 (severe) for each parameter¹⁵. The maximum mean histopathological damage score was 15. Tissue sections were examined with a Leica DFC 280 light microscope and Leica Q Win image analysis system (Leica Microscope Imaging Solution Ltd, Cambridge, UK) and evaluated, and their photographs were taken.

Immunohistochemical Analysis

Immunohistochemical (IHC) staining was performed using caspase-3 antibody (ab13847; Abcam, Kimera, Turkey). Tubular and glomerular caspase-3 immunoreactions were examined semi-quantitatively under a Leica DFC 280 light microscope. To determine the staining intensity, 10 fields from each section were examined at X20 magnification and scored as (0) no staining, (1) weak staining, (2) moderate staining, and (3) severe staining. Tissue sections were examined with Leica DFC 280 light microscope and Leica Q Win image analysis system, scored and photographed.

Statistical Analysis

Statistical analyses were performed with SPSS (SPSS for Windows version 13.0) software. All results were expressed as arithmetic mean±standard error. In comparison of the groups, the Kruskal Wallis analysis of variance, which is one of the non-parametric tests, was used to compare all groups for all variables, while the Mann-Whitney U test was used for pairwise comparison of variables. A p value of <0.05 was considered statistically significant.

RESULTS

Histopathological Findings

The control group had normal histological appearance. The quercetin group had normal histological appearance like the control group. There was no statistically significant difference between the control group and the quercetin group in terms of histopathological findings (p>0.05). The mean histopathological damage score in the cisplatin group (Figure 1F) was statistically significantly increased compared to the control (Figure 1A) and quercetin (Figure 1B) groups (p=0.001). In the cisplatin group, dilatation of tubules, epithelial shedding into tubules (tubular caste), swelling and vacuolization of tubular epithelial cells and loss of microvillus in proximal tubules were detected (Figure 1C, 1E, 1H). In addition, increased PAS (+) staining intensity in the glomeruli, extensive hemorrhage, and occasionally peritubular infiltration areas were also observed in the cisplatin group (Figure 1D, 1F, 1G, 1H). All histopathological findings evaluated in the Cisplatin+Quercetin group were significantly reduced compared to the cisplatin group (p=0.001) (Table 1) (Figure 1C-K). The mean histopathological damage scores of all groups are given in Table 1.

Immunohistochemical Findings

Mild staining was detected in the control and quercetin groups with anti-Caspase-3 antibody (Figures 2A, 2B). In the cisplatin group, the intensity of the staining was noticeably increased, especially in the proximal tubules. When the Cisplatin and Cisplatin+Quercetin groups were compared, a statistically significant decrease in Caspase-3 IHC staining intensity was observed (p<0.005) (Figure 2C, 2D). The IHC scores of all groups are given in Table 2.

DISCUSSION

One of the most common side effects of cisplatin is dose-dependent renal toxicity¹⁶. Cisplatin nephrotoxicity may present with very different manifestations such as acute kidney injury, distal renal acidosis, hyperuricemia, hypomagnesemia, hypocalcemia or chronic kidney failure^17-22. Cisplatin nephrotoxicity is the result of transport of cisplatin to renal epithelial cells, injury of nuclear and mitochondrial DNA, activation of cell death pathways, and initiation of a strong inflammatory response²³.

In a study, tubular degeneration and necrosis, hyaline eruptions in the tubules, intertubular hemorrhage, glomerular obstruction, and vacuolization were reported in the kidneys of male Sprague-Dawley rats weighing 200 grams that were administered a single intraperitoneal dose of 7 mg/kg cisplatin²⁴. We also applied the same dose of cisplatin in our study and observed the findings of dilatation and epithelial shedding (tubular caste) in the renal tubules. In another recent study, perivascular inflammatory cell infiltration, as well as tubular vacuolar degeneration and hyaline desquamation in the lumen of the tubules, was detected in the kidneys of rats administered the same dose of cisplatin as ours²⁵. These findings also support our study.

In a study conducted to observe the antioxidant effects against cisplatin renal toxicity, microvillus deformation in the tubules and focal loss were observed in electron microscopic imaging of kidney tissue²⁶. In our study, we observed loss of microvillus in the proximal tubules in sections with PAS staining.

Due to the serious side effects of cisplatin, various agents with antioxidant activity are being tested with this chemotherapeutic drug²⁷. Quercetin is one of the most common dietary polyphenolic compounds, which is abundant in many foods. It is an effective antioxidant against radical oxygen species that prevents oxidation of low-density lipoproteins by scavenging free radicals and chelating transition metal ions^6,28. It has anti-inflammatory, anticarcinogenic and antiviral properties^29-31.

In a study conducted to reverse the effects of nephrotoxicity, it was observed that histopathological findings such as renal tubular degeneration caused by lead element, necrosis, vacuolization and mononuclear cell infiltration regressed with 10 mg/kg quercetin and returned to normal histological appearance³². In our study, we found that dilatation in the renal tubules and vacuolization in tubular epithelial cells improved in the Cisplatin+quercetin group compared to the cisplatin group.

In renal damage induced by cyclosporine, quercetin has been found to reduce the findings of interstitial fibrosis, arteriopathy, glomerular basement membrane thickening, vacuolization of tubular epithelial cells and desquamation into the tubular lumen³³. In our study, we observed that epithelial shedding (tubular caste) and vacuolization findings decreased with the application of quercetin.

In a study investigating the effects of quercetin in a diabetic nephropathy model, it was reported that histopathological findings such as epithelial desquamation, intracytoplasmic vacuolization, loss of brush border in the proximal tubules, and peritubular infiltration were reduced by the administration of quercetin¹⁵. In our study, we found that tubular dilatation, epithelial shedding in the tubule lumen (tubular caste), vacuolization in tubular epithelial cells, and loss of microvilli in the proximal tubules improved with the application of quercetin.

Caspase-3 is a key zymogen in cell apoptosis⁹. In a study, rats with experimental mammary adenocarcinoma were administered 4 mg/kg cisplatin and then 50 mg/kg quercetin. It has been observed that cisplatin causes shedding of renal tubular epithelial cells, loss of brush border, hyaline deposition in the tubule lumen, and enlargement of tubules. It has been reported that as a result of caspase-3 immunostaining, the staining intensity increased with cisplatin, but the staining intensity decreased in the cisplatin+quercetin group³⁴. In our study, 7 mg/kg cisplatin and 25 mg/kg quercetin were administered and it was determined that the staining intensity increased with caspase-3 antibody.

Study Limitations

Although the current study has given the expected results, it has some limitations. The most important limitation is that there had to be biochemical data because quercetin is an antioxidant substance. In addition, validation of data obtained from examinations with electron microscopy can make the results even more reliable.

TARTIŞMA

Sisplatinin en yaygın yan etkilerinden biri doza bağımlı renal toksisitedir¹⁶. Sisplatin nefrotoksisitesi akut böbrek hasarı, distal renal asidoz, hiperürisemi, hipomagnezemi, hipokalsemi veya kronik böbrek yetmezliği gibi çok farklı tablolarla karşımıza çıkabilir^17-22. Sisplatin nefrotoksisitesi sisplatinin renal epitel hücrelerine taşınması, nükleer ve mitokondriyal DNA’nın yaralanması, hücre ölümünün yollarının aktivasyonu ve güçlü bir inflamatuvar yanıtın başlamasının sonucudur²³.

Yapılan bir çalışmada, intraperitoneal tek doz 7 mg/kg sisplatin uygulanan erkek Sprague-Dawley cinsi 200 gram ağırlığında sıçanların böbreklerinde tubuler dejenerasyon ve nekroz, tubullerde hiyalin döküntüleri, intertubuler kanama, glomerülde tıkanıklık ve vakuolizasyon bildirilmiştir²⁴. Biz de çalışmamızda aynı doz sisplatin uyguladık ve renal tubullerde dilatasyon, epitelyal dökülme (tubular kast) bulgularını gözlemledik. Yakın zamanda yapılan başka bir çalışmada, yine bizimle aynı doz sisplatin uygulanan sıçanların böbreklerinde, tubuler vakuolar dejenerasyon ve tubullerin lümeninde hiyalin deskuamasyonun yanı sıra perivasküler enflamatuvar hücre infiltrasyonu da tespit edilmiştir²⁵. Bu bulgular da çalışmamızı desteklemektedir.

Sisplatin renal toksisitesine karşı antioksidan etkileri gözlemlemek için yapılan bir çalışmada, böbrek dokusunun elektron mikroskobik görüntülenmesinde tubullerde mikrovillus yapısının bozulduğu ve fokal kaybı gözlenmiştir²⁶. Çalışmamızda, PAS boyaması yapılan kesitlerde proksimal tubullerde mikrovillus kaybını gözlemledik.

Sisplatinin ciddi yan etkileri sebebiyle çeşitli antioksidan etkinliği olan ajanlar bu kemoterapötik ilaçla birlikte denenmektedir²⁷. Kuersetin birçok besinde bol miktarda bulunan en yaygın diyet polifenolik bileşiklerinden biridir. Serbest radikalleri temizleyerek ve geçiş metal iyonlarını şelatlayarak düşük yoğunluklu lipoproteinlerin oksidasyonunu önleyen radikal oksijen türlerine karşı etkili bir antioksidandır^6,28. Anti-enflamatuvar, anti-kanserojenve antiviral özelliklere sahiptir^29-31.

Nefrotoksisitenin etkilerinin geri döndürülmesi amacıyla yapılan bir çalışmada, kurşun elementinin oluşturduğu renal tubuler dejenerasyon, nekroz, vakuolizasyon ve mononükleer hücre infiltrasyonu gibi histopatolojik bulguların 10 mg/kg kuersetin ile gerilediği ve normal histolojik görünüme döndüğü gözlenmiştir³². Çalışmamızda, sisplatin+kuersetin grubumuzda, sisplatin grubuna göre renal tubullerde dilatasyonun ve tubul epitel hücrelerinde vakuolizasyonun düzeldiğini tespit ettik.

Siklosporin ile oluşturulan renal hasarda kuersetinin interstisyel fibrozis, arteriyopati, glomerüler bazal membran kalınlaşması, tubuler epitel hücrelerinde vakuolizasyon ve tubul lümenine deskuamasyon bulgularını azalttığı tespit edilmiştir³³. Yaptığımız çalışmamızda kuersetin uygulaması ile epitelyal dökülme (tubular kast) ve vakuolizasyon bulgularının azaldığını gözlemledik.

Diyabetik nefropati modelinde kuersetinin etkilerinin araştırıldığı bir çalışmada epitelyal deskuamasyon, intrasitoplazmik vakuolizasyon, proksimal tubullerde fırçamsı kenar kaybı ve peritübüler infiltrasyon gibi histopatolojik bulguların kuersetin uygulamasıyla azaldığı bildirilmiştir¹⁵. Çalışmamızda sisplatin ile oluşan tubuler dilatasyon, tubul lümenine epitelyal döküntü (tubular kast), tubul epitel hücrelerinde vakuolizasyon ve proksimal tubullerde mikrovillus kaybının kuersetin uygulaması ile düzeldiğini tespit ettik.

Caspase-3, hücre apoptozunda anahtar bir zimojendir⁹. Yapılan bir çalışmada, deneysel meme adenokarsinomu oluşturalan sıçanlara 4 mg/kg sisplatin ve ardından için 50 mg/kg kuersetin uygulanmıştır. Sisplatinin renal tubuler epitel hücrelerinde dökülme, fırçamsı kenar kaybı, tubul lümeninde hiyalin birikimi ve tubullerin genişlemesine yol açtığı görülmüştür. Caspase-3 immün boyaması sonucunda, sisplatin ile boyama yoğunluğunun arttığı, fakat sisplatin+kuersetin grubunda boyanma yoğunluğunun azaldığı bildirilmiştir³⁴. Çalışmamızda, 7 mg/kg sisplatin ve 25 mg/kg kuersetin uygulanmış ve caspase-3 antikoru ile boyanma yoğunluğunun arttığı tespit edilmiştir.

Çalışmanın Kısıtlılıkları

Sunulan çalışma, düşündüğümüz sonuçları ortaya koymasının yanı sıra, bazı sınırlamalara sahiptir. En önemli sınırlama, Kuersetinin antioksidan bir madde olması sebebiyle biyokimyasal verileri olmalıydı. Ek olarak elektron mikroskobi ile incelemelerden elde edilen verilerin doğrulanması sonuçları daha da güvenilir hale getirebilir.

CONCLUSION

As a result, it is seen that quercetin is histopathologically beneficial in the treatment of kidney toxicity caused by cisplatin and has a positive effect on apoptotic pathways. We think that further studies on this subject with different doses and durations will contribute to the literature.