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    Expression patterns and distribution of aquaporin water channels in cervix as a possible mechanism for cervical patency in bitches affected by pyometra
    (Elsevier Science Inc, 2024) Yazlik, Murat Onur; Ozkan, Hueseyin; Vural, Sevil Atalay; Kaya, Ufuk; Ozoner, Ozgur; Mutluer, Ipek; Altinbas, Yunus Furkan
    Pyometra is a life-threatening disease, the severity of which depends on cervical patency status. This study investigated cervical inflammation status as well as the expression patterns and localization of aquaporin (AQP1, AQP2, AQP3, AQP5, and AQP9), and hormone receptors in cervical tissue that influences canine pyometra. Of the 36 animals enrolled in the study, 24 were diagnosed with pyometra and separated into two groups: open cervix pyometra and close cervix pyometra, while 12 healthy animals presented for elective ovariohysterectomies were allocated into the control group. Surgical treatment was performed for treatment of pyometra. After each operation, cervix samples were collected and analyzed for AQP and hormone receptor expression patterns determined by qPCR and protein expression by means of immunohistochemistry. Blood samples were also collected to determine serum progesterone concentrations. AQP9 expression was downregulated approximately 3-fold while and PGR expression was downregulated more than 2 fold in both pyometra groups compared to the control group. AQP3 and AQP5 gene expression levels were upregulated more than 3 fold in the open-cervix pyometra group than the closed-cervix pyometra group (P < 0.05). This is the first study to describe the expression patterns and immunolocalization of AQPs in canine cervical tissue based on pyometra patency status and to report AQP3 and AQP5 expression in cervical tissue linked to cervical patency.
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    Öğe
    Pyometra alters uterine aquaporins related with lipopolysaccharide concentrations and antioxidant enzyme activities in bitches
    (Elsevier, 2024) Yazlik, Murat Onur; Ozkan, Huseyin; Vural, Sevil Atalay; Kaya, Ufuk; Mustalk, Inci Basak; Altinbas, Ipek Mutluer; Tunc, Arda Selin
    Pyometra is a common life-threatening inflammatory disease with a complex etiopathogenesis that develops during the diestrus stage and can be observed in elderly intact bitches. The present study evaluated five aquaporin (AQP1, AQP2, AQP3, AQP5, and AQP9) transcript abundances and immunolocalization in the uterine tissue, and investigated their relationship with uterine tissue and blood lipopolysaccharide (LPS) concentration, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity, and nitric oxide (NO) production in dogs suffering from pyometra. The study sampled 36 client-owned intact bitches from different breeds, of which 24 cases were diagnosed with pyometra. Twelve of these bitches in the diestrus stage that presented for elective ovariohysterectomy were used as the control group. Blood samples were collected into tubes without anticoagulant for serum progesterone, LPS concentration, and antioxidant activities at the time of diagnosis. Bacteriological and tissue samples from the uteri were collected after the ovariohysterectomy. The tissue samples were used to determine antioxidant activity, and hor- mone and toxin concentrations. Transcript abundance of uterine AQPs were determined by qPCR, and their presence and localization were determined by by immunohistochemistry. For all pyo- metra samples, the bacteria isolated from the uterine swabs were Escherichia coli. Compared to the control group, AQP1, AQP2, and AQP5 were downregulated more than 2-fold, whereas AQP9 was upregulated nearly 3-fold and AQP3 was upregulated more than 4-fold in the pyometra affected uteri (P<0.05). Uterine AQP1 was moderately negatively correlated with serum LPS concentra- tion (r=-0.568, P<0.01) and tissue NO production (r=-0.407, P<0.05). AQP5 was positively correlated with serum SOD activity (r=0.485, P<0.05) and negatively correlated with serum LPS concentration (r=-0.512, P<0.05). AQP9 was negatively correlated with tissue SOD and serum GPx activity. This is the first study to identify AQP9 transcript abundance and immunolocaliza- tion in canine uterine tissue. Uterine AQP1, AQP2, AQP3, AQP5, and AQP9 transcript abundances were altered in spontaneously developed canine pyometra while AQP transcript abundance was negatively related to serum toxin concentration, NO production, and antioxidant enzyme activity. Further studies should be conducted to determine the role of altered abundances of AQPs tran- scripts in pyometra pathogenesis.
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    Öğe
    Spontan Gelişen Köpek Pyometralarında Aquaporinlerin Rolü ve Ovaryum Steroidleri, Antioksidan Enzim Aktiviteleri ile İlişkisinin Ortaya Konulması
    (2023) Yazlık, Murat Onur; Vural, Mehmet Rıfat; Vural, Sevil; Tunç, Arda Selin; Müştak, İnci Başak; Kaya, Ufuk; Özöner, Özgür
    Pyometra, köpeklerde diöstrus döneminde gözlenen genellikle ölümle seyreden yangısal bir hastalıktır. On yaşından daha genç köpeklerin ortalama %19?unda gözlenebilmektedir. Multifaktöriyel bir etiyolojiye sahip olduğundan bütün etiyopatogeneziste açıklanamayan mekanizmalar bulunmaktadır. Bu projede yangısal faktörlerin düzenlenmesinde de görev alan aquaporin (AQP)?lerin Escherichia coli (E. coli) nedenli oluşan açık ve kapalı serviks pyometra olgularında ovaryum, uterus ve serviks dokusundaki gen ekspresyon düzeylerinin siklusun anöstrus ve diöstrus dönemindeki sağlıklı köpeklere kıyasla değişimleri belirlenmiştir. Ayrıca, sağlıklı ve pyometralı köpeklerde dokudaki AQP ekspresyonları ile serum ve doku superoksid dismutaz (SOD), glutatione peroksidaz (GPx) gibi antioksidan enzimler ile nitrik oksit (NO) ve lipopolisakkarit (LPS) düzeyleri, serum progesteron düzeyi ve lokal hormon reseptör ekspresyonları ile ilişkisi belirlenmiştir. Dahası enfeksiyona neden olan E.coli virulens gen dağılımlarına göre uterus dokusundaki AQP ekspresyon değişimlerinin belirlenmesi amaçlanmıştır. Çalışmaya toplamda 48 adet dişi köpek dahil edilmiştir. Kontrol grubunu rutin kısırlaştırma amacıyla kliniğe getirilen sağlıklı 24 köpek oluşturmuştur (Diöstrus dönemi n:12; Anöstrus dönemi n:12). Çalışmanın deney grubunu E.coli nedenli pyometradan muzdarip köpekler oluşturmuştur (Açık serviks n:12; Kapalı serviks n:12). Çalışmaya dahil edilen köpeklerin tamamına rutin ovariyohisterektomi operasyonu gerçekleştirilmiştir. Bakteriyoloji, ovaryum, uterus ve serviksteki gen ekspresyonları ile kan ve uterus dokusundaki enzim ve toksin düzeylerini amacıyla örnekler toplanmıştır. Doku örnekleri histopatolojik ve immunohistokimyasal incelemelerden geçirilerek doku düzeyinde AQP ve lokal hormon reseptörleri antijenlerinin varlığı semikantitatif olarak belirlenmiştir. Gen ekspresyon düzeylerini belirlemek için toplanan örnekler RNA later solüsyonuna konularak -80°C?de analiz gününe kadar saklanmıştır. Total RNA izolasyonu modifiye edilmiş TRIzol metoduna göre yapılmıştır. Köpeklerde ovaryum, uterus ve serviks dokularında ER, PGR, OTR, AQP1, AQP2, AQP3, AQP5 ve AQP9 genlerinin ekspresyon seviyelerinin araştırılması amaçlanmıştır. Bu amaçla GAPDH ve RPS15 housekeeping genleri kullanılmış ve Primer-Blast (NCBI) ile Primer 3 Plus programları aracılığıyla köpek türünde hedef genlerin amplifikasyonu için spesifik Forward ve Reverse primer dizileri tasarlanmıştır. Real Time PCR?da hedef genler örneklerde 3 tekrarlı olacak şekilde çalışılmıştır. Genlerin amplifikasyonunda SYBR Green I boya içeren kit kullanılmıştır. Virülens genlerin belirlenmesi amacıyla konvansiyonel PCR yöntemi kullanıldı. Serum progesteron, LPS, SOD, GPx ve NO düzeyleri antikoagulansız tüplere alınan kan örneklerinin santrifüjü sonrası elde edilen serumdan ölçülmüştür. Doku ve serumdaki antioksidan aktivite, toksin düzeyleri ve serum progesteron değerleri ticari ELİSA kitleri kullanılarak belirlenmiştir. Pyometra olgularında ovaryum, uterus ve serviks dokusunda AQP ekspresyon seviyelerinin değişim gösterdiği; pyometraların açık veya kapalı serviks tipinde olmasına göre uterus dokusunda AQP ekspresyonlarının farklılaştığı; izole edilen E.coli?lerde virülens genlerin bulunması durumunda AQP?lerin uterus dokusundaki ekspresyonlarında farklılıklar gözlenebileceği; pyometralı köpeklerde kanda artan LPS düzeyinin uterus AQP ekspresyonlarıyla ilişkili olduğu; köpeklerde pyometra olgularında serviksin açılma mekanizmasında AQP?lerin rol alabileceği; SOD, GPx ve NO düzeylerinin uterus ve serviks dokusundaki bazı AQP ekspresyonlarından ile ilişkili olduğu belirlenmiştir.
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    Öğe
    The presence of virulence factor genes downregulates uterine AQP3 and alters glutathione peroxidase activity and uterine histopathology in canine pyometra
    (Wiley, 2024) Yazlik, Murat Onur; Mustak, Inci Basak; Ozkan, Hueseyin; Vural, Sevil Atalay; Kaya, Ufuk; Ozoner, Ozgur; Mutluer, Ipek
    Present study was designed to evaluate the role of virulence factor genes (papG, cnf1 and hylA) in the pathogenesis of canine pyometra. Antimicrobial susceptibility test and detection of virulence genes were performed Escherichia coli (E. coli) detected in uterine swab samples. Animals were divided into two groups based on the presence (VF+, n:14) or absence (VF-, n:7) of the virulence factor genes papG, cnf1 and hylA. Blood and tissue glutathione peroxidase activity, uterine histopathologic analysis and AQP3, ESR1, PGR, OXTR gene expressions were determined in both groups. Statistical analyses were performed using Stata version 15.1. All E. coli isolates were susceptible to amikacin, whereas resistant to ampicillin, amoxicillin/clavulanic acid and lincomycin. None of the isolates were susceptible to cefotaxime. E. coli isolates had at least one virulence gene. The most prevalent gene was fimH (100%), followed by fyuA (95.8%), usp (83.3%), sfa (75%), cnf1 and hlyA (70.8%) genes. Blood GPx activity was greater in VF+ animals. On the other hand, uterine tissue GPx activity was lower in VF+ group compared to the control group. Expression levels of AQP3 were upregulated more than fivefold in VF-dogs compared to the control group. In addition, AQP3 expression levels were found approximately threefold higher in VF (-) than VF (+) group (p < .05). Varying degree of inflammation noted for all animals with pyometra, but the presence of bacteria noted only in VF+ animals. In conclusion, the presence of virulence factor genes does not play a role in the histopathological degree of inflammation, the presence of bacteria was found to vary. Serum GPx activity increased in VF+ animals. While the hormone receptor expressions were similar, AQP expression was upregulated in the absence of virulence factor genes.