Osteopontin synthesis and localization in different cultured renal cells

Abstract

Osteopontin (OPN) is a glycoprotein which synthesized within the kidney and secreted into the urine by epithelial cells and can inhibit forming the kidney stones especially calcium oxalate stones. The aim of this study is to compare levels of OPN in MDCK (Madin Darby Canine Kidney) cells with different culture conditions and levels of secreted OPN in different culture conditions. Another objective of this study is determination of OPN localization in MDCK cells with different culture conditions. MDCK II (Madin Darby Canine Kidney) cell line were used. OPN expression in MDCK cells was investigated with RT-PCR and agar gel electrophoresis methods. MDCK cells treated with NaHPO4, MgSO4, CaCl2, Na3C6H5O7 and COM, intracellular distribution of osteopontin in that cells were monitored by confocal immunofluorescence microscopy tecnique. Intracellular OPN levels were analysed by Western blot. Secreted OPN in the medium were also analysed by Western blot after immunoprecipitation. Osteopontin levels in different MDCK culture conditions and secreted in extracellular matrix were compared with control groups, there was found any differences between the groups. Intracellular homogen distribution of OPN in cells that made with Na3C6H5O7 and COM solutions were not monitored, osteopontin conglomerates near the cell membrane as a fibrillar aggregate were observed in that cells. Intracellular localization of OPN in different MDCK cell culture conditions was changed. In order to explain functions of OPN, it is necessary that making more researches about structures and modifications of OPN isoforms

Keywords

• Calcium oxalate
• MDCK cell line
• osteopontin
• RT-PCR
• Western blot

Farklı kültür ortamlarındaki böbrek hücrelerinde osteopontin sentezi ve lokalizasyonu

Abstract

Osteopontin (OPN), böbrek epitel hücrelerinden idrara salgılanan ve üriner sistemde taş oluşumuna karşı (özellikle kalsiyum okzalat taşı oluşumunda) savunma mekanizmasında önemli rolü olan bir glikoproteindir. Bu çalışmada, OPN’nin taş oluşumunu indükleyen farklı kültür ortamlarındaki MDCK (Madin Darby Canine Kidney) hücrelerinde ve ortama salınan düzeylerindeki değişikliklerin incelenmesi amaçlanmıştır. Çalışmanın diğer bir amacı ise farklı kültür ortamlarındaki MDCK hücrelerinde OPN’in lokalizasyonunda oluşabilecek değişikliklerin araştırılmasıdır. Çalışmada MDCK II (Madin Darby Canine Kidney) hücre hattı kullanılmıştır. MDCK hücrelerinde OPN ekspresyonu, RT-PCR takiben agar jel elektroforez ile gösterilmiştir. NaHPO4, MgSO4, CaCl2, Na3C6H5O7 ve NaOx/CaCl2 (COM) uygulamasıyla oluşturulan farklı kültür ortamlarındaki osteopontinin hücre içi yerleşimi immunofloresans konfokal mikroskopla gösterilmiştir. Hücre içi OPN düzeyleri Western blot yöntemiyle, ortama salgılanan OPN düzeyleri ise immunopresipitasyon sonrası Western blot ile gösterilmiştir. Farklı kültür ortamlarındaki MDCK hücrelerinde ve ekstraselüler matrikse salınan OPN düzeylerinde kontrol grubuna göre herhangi bir farklılık tespit edilememiştir. Na3C6H5O7 ve COM eklenen gruplarda konfokal lazer mikroskobik incelemesinde OPN’in hücre içi homojen dağılımının bozulduğu ve fibriler kümelenmeler tarzında hücre zarına doğru biriktiği gözlenmiştir. Farklı MDCK kültür ortamlarında, OPN’in hücre içi lokalizasyonunda değişiklikler şekillenmiştir. OPN’in fonksiyonlarını açıklamak için, OPN izoformlarının yapısı ve modifikasyonlarıyla ilgili daha çok araştırmaya gerek duyulmaktadır

Keywords

• Kalsiyum okzalat
• MDCK hücre hattı
• osteopontin
• RT-PCR
• Western blot

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