Öz
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
Anahtar Kelimeler
Calcium oxalate MDCK cell line osteopontin RT-PCR Western blot
Kaynakça
- Agnihotri R, Crawford HC, Haro H ve ark. (2001): Osteopontin, a novel substrate for matrix metalloproteinase- 3 (stromelysin-1) (matrilysin). J Biol Chem, 276, 28261-28267. matrix metalloproteinase-7
- Altıntaş A, Saruhan G, Benbir G ve ark. (2009): The role of osteopontin: A Shared pathway in the pathogenesis of multiple sclerosis and osteoporosis. J Neurol Sci, 276, 41-44.
- Cha J, Ahn Y, Lim SW ve ark. (2000): Decreased osteopontin expression in the rat kidney on a sodium deficient diet. YMJ, 41, 128-135.
- Chien Y, Masica DL, Gray JJ ve ark. (2009): Modulation of calcium oxalate dihydrate growth by selective crystal- face polyaspartate peptide showing occlusion by sectoral (compositional) zoning. JBC, 284, 23491-23501. and
- Denhardt DT, Xiaojia G (1993): Osteopontin: A protein with diverse functions. FASEB J, 7, 1475-1482.
- Gericke A, Qin C, Spevak L ve ark. (2005): Importance of phosphorlylation for osteopontin regulation of biomineralization. Calcif Tissue Int, 77, 45-54.
- Giachellia CM, Steitz S (2000): Osteopontin: A versatile regulator of inflammation an biomineralization. Matrix Bio, 19, 615-622.
- Gilbert M, Shaw WJ, Long JR ve ark. (2000): Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion. J Biol Chem, 275, 16213-16218.
- Hoyer JR, Asplin JR, Otvos L (2001): Phosphorylated osteopontin peptides suppres crystallization by inhibition the growth of calcium oxalate crystals. Kidney Int, 60, 77-82.
- Hudkins KL, Giachelli CM, Cui Y ve ark. (1999): Osteopontin expression in fetal and mature human kidney. J Am Soc Nephrol, 10, 444-457.
- Hunter GK, Kyle CL, Goldberg HA (1994): Modulation of crystal formation by bone phosphoproteins: Structural specificity of the osteopontin-mediated inhibition of hydroxypatite formation. Biochem J, 300, 723-728.
- Itoh Y, Yasui T, Okada A ve ark. (2005): Examination of the antioxidative effect in renal tubular cells and apoptosis by oxidative stres. Urol Res, 33, 261-266.
- Kohri K, Nomura S, Kitamura Y ve ark. (1993): Structure and expression of the mRNA encoding urinary stone protein (osteopontin). J Biol Chem, 20, 15180-15184.
- Kumar V, Vega LP, Farell G ve ark. (2005): Urinary macromolecular inhibition of crystal adhesion to renal epithelial cells is impaired in male stone formers. Kidney Int, 68, 1784-1792.
- Langdon A, Wignall GR, Rogers K ve ark. (2009): Kinetics of calcium oxalate crystal growth in the presence of osteopontin isoforms: an analysis by scanning confocal interference microcopy. Calcif Tissue Int, 84, 240-248.
- Lieske JC, Hammes MS, Hoyer JR ve ark. (1997): Renal cell osteopontin production is stimulated by calcium oxalate monohydrate crystals. Kidney Int, 51, 679-686.
- Liu Y, Zhang D, Sui X ve ark. (2009): Overexpression of human osteopontin increases cell proliferation and migration in human embryo kidney-293 cells. Cell Mol Biol Let, 14, 670-678.
- Mazzali M, Kipari T, Ophascharoensk V ve ark. (2002): Osteopontin-a molecule for all seasons. Q J Med, 95, 3-13.
- Min W, Shiraga H, Chalko C ve ark. (1998): Quantitative studies of human urinary excretion of uropontin. Kidney Int, 53, 189-193.
- O’Young J, Chirico S, Tarhuni N ve ark. (2009): Phosphorylation adsorption to and incorporation into calcium oxalate crystals. Cells Tissues Organs, 189, 51-55. mediates
- Peracchia C (1977): Calcium effects on gap junction structure and cell coupling. Nature, 271, 669-671.
- Qui SR, Wierzbicki A, Orme CA ve ark. (2007): Molecular modulation of calcium oxalate crystallization by osteopontin and citrate. PNAS, 101, 1811-1815.
- Reynolds JA, Tanford C (1970): The gross conformation of protein-sodium dodecyl sulfate complexes. J Biol Chem, 245, 5161-5165.
- Robinovich YI, Esayanur M, Daosukho S ve ark. (2006): Adhesion force between calcium oxalate monohydrate crystal and kidney epithelial cells and possible relevance for kidney stone formation. J Coll Inter Sci, 300, 131-140.
- Silverman L, Boskey AL (2004): Diffusion systems for evaluation of biomineralization. Calcif Tissue Int, 75, 494-501.
- Sodek J, Ganss B, Mckee MD (2000): Osteopontin. Crit Rev Oral Biol. Med, 11, 279-303.
- Sun BS, You J, Li Y ve ark. (2013): Osteopontin knockdown suppresses non small cell lung cancer cell invasion and metastasis. Chin Med J, 126, 1683-1688.
- Svennevig K, Prydz K, Kolset SO (1995): Proteoglycans in polarized epithelial Madin-Darbycanine kidney cells. Biochem J, 311, 881-888.
- Taller A, Grohe B, Rogers KA ve ark. (2007): Specific adsorption of osteopontin and synthetic polypeptides to calcium oxalate monohydrate crystals. Biophys J, 93, 1768-1777.
- Taşçene N, İsgüder Z, Salmanoğlu B (2014): Osteopontin expression in polarized MDCK cells. Kafkas Univ Vet Fak Derg, 20, 671-674.
- Ulrich O, Mann K, Haase W ve ark. (1991): Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line. J Biol Chem, 266, 3518-3525.
- Umekawa T, Iguchi M, Uemura H ve ark. (2006): Oxalate ions and calcium oxalate crystal-induced up- regulation of osteopontin and monocyte chemoattractant protein-1 in renal fibroblasts. BJU Int, 98, 656-660.
- Verhulst A, Persy VP, Rompay AV ve ark. (2002): Osteopontin synthesis and localization along the human nephron. J Am Soc Nephrol, 13, 1210-1218.
- Wang L, Guan X, Tang R ve ark. (2008): Phosphorylation of osteopontin is required for inhibition of calcium oxalate crystallization. J Phys Chem B, 112, 9151-9157.
- Wesson JA, Johnson RJ, Mazzali M ve ark. (2003): Osteopontin is a critical inhibitör of calcium oxalate crystal formation and retention in renal tubules. J Am Soc Nephrol, 14, 139-47.
- Xie Y, Sakatsume M, Nishi S ve ark. (2001): Expression, roles, receptors and regulation of osteopontin in the kidney. Kidney Int, 60, 1645-1657.
- Yamaguchi S, Wiessner J, Hasegawa A ve ark. (2002): Calcium oxalate monohydrate crystal binding substance produced from Madin-Darby canine kidney cells. Int J Urol, 9, 50-58.
- Yamate T, Kohri K, Umekawa T ve ark. (1998): Interaction between osteopontin on Madin-Darby canine kidney cell membrane and calcium oxalate crystal. Urol Int, 62, 81-86.
- Yamate T, Tsuji H, Amasaki N ve ark. (2000): Analysis of osteopontin DNA in patients with urolithiasis. Urol Res, 28, 159-166.
- Yasui T, Sato M, Fujıta K ve ark. (2001): Effects of citrate on renal stone formation and osteopontin in a rat urolithiasis model. Urol Res, 29, 50-56. Geliş tarihi: 01.07.2015 / Kabul tarihi: 09.08.2016
Öz
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
Anahtar Kelimeler
Kalsiyum okzalat MDCK hücre hattı osteopontin RT-PCR Western blot
Kaynakça
- Agnihotri R, Crawford HC, Haro H ve ark. (2001): Osteopontin, a novel substrate for matrix metalloproteinase- 3 (stromelysin-1) (matrilysin). J Biol Chem, 276, 28261-28267. matrix metalloproteinase-7
- Altıntaş A, Saruhan G, Benbir G ve ark. (2009): The role of osteopontin: A Shared pathway in the pathogenesis of multiple sclerosis and osteoporosis. J Neurol Sci, 276, 41-44.
- Cha J, Ahn Y, Lim SW ve ark. (2000): Decreased osteopontin expression in the rat kidney on a sodium deficient diet. YMJ, 41, 128-135.
- Chien Y, Masica DL, Gray JJ ve ark. (2009): Modulation of calcium oxalate dihydrate growth by selective crystal- face polyaspartate peptide showing occlusion by sectoral (compositional) zoning. JBC, 284, 23491-23501. and
- Denhardt DT, Xiaojia G (1993): Osteopontin: A protein with diverse functions. FASEB J, 7, 1475-1482.
- Gericke A, Qin C, Spevak L ve ark. (2005): Importance of phosphorlylation for osteopontin regulation of biomineralization. Calcif Tissue Int, 77, 45-54.
- Giachellia CM, Steitz S (2000): Osteopontin: A versatile regulator of inflammation an biomineralization. Matrix Bio, 19, 615-622.
- Gilbert M, Shaw WJ, Long JR ve ark. (2000): Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion. J Biol Chem, 275, 16213-16218.
- Hoyer JR, Asplin JR, Otvos L (2001): Phosphorylated osteopontin peptides suppres crystallization by inhibition the growth of calcium oxalate crystals. Kidney Int, 60, 77-82.
- Hudkins KL, Giachelli CM, Cui Y ve ark. (1999): Osteopontin expression in fetal and mature human kidney. J Am Soc Nephrol, 10, 444-457.
- Hunter GK, Kyle CL, Goldberg HA (1994): Modulation of crystal formation by bone phosphoproteins: Structural specificity of the osteopontin-mediated inhibition of hydroxypatite formation. Biochem J, 300, 723-728.
- Itoh Y, Yasui T, Okada A ve ark. (2005): Examination of the antioxidative effect in renal tubular cells and apoptosis by oxidative stres. Urol Res, 33, 261-266.
- Kohri K, Nomura S, Kitamura Y ve ark. (1993): Structure and expression of the mRNA encoding urinary stone protein (osteopontin). J Biol Chem, 20, 15180-15184.
- Kumar V, Vega LP, Farell G ve ark. (2005): Urinary macromolecular inhibition of crystal adhesion to renal epithelial cells is impaired in male stone formers. Kidney Int, 68, 1784-1792.
- Langdon A, Wignall GR, Rogers K ve ark. (2009): Kinetics of calcium oxalate crystal growth in the presence of osteopontin isoforms: an analysis by scanning confocal interference microcopy. Calcif Tissue Int, 84, 240-248.
- Lieske JC, Hammes MS, Hoyer JR ve ark. (1997): Renal cell osteopontin production is stimulated by calcium oxalate monohydrate crystals. Kidney Int, 51, 679-686.
- Liu Y, Zhang D, Sui X ve ark. (2009): Overexpression of human osteopontin increases cell proliferation and migration in human embryo kidney-293 cells. Cell Mol Biol Let, 14, 670-678.
- Mazzali M, Kipari T, Ophascharoensk V ve ark. (2002): Osteopontin-a molecule for all seasons. Q J Med, 95, 3-13.
- Min W, Shiraga H, Chalko C ve ark. (1998): Quantitative studies of human urinary excretion of uropontin. Kidney Int, 53, 189-193.
- O’Young J, Chirico S, Tarhuni N ve ark. (2009): Phosphorylation adsorption to and incorporation into calcium oxalate crystals. Cells Tissues Organs, 189, 51-55. mediates
- Peracchia C (1977): Calcium effects on gap junction structure and cell coupling. Nature, 271, 669-671.
- Qui SR, Wierzbicki A, Orme CA ve ark. (2007): Molecular modulation of calcium oxalate crystallization by osteopontin and citrate. PNAS, 101, 1811-1815.
- Reynolds JA, Tanford C (1970): The gross conformation of protein-sodium dodecyl sulfate complexes. J Biol Chem, 245, 5161-5165.
- Robinovich YI, Esayanur M, Daosukho S ve ark. (2006): Adhesion force between calcium oxalate monohydrate crystal and kidney epithelial cells and possible relevance for kidney stone formation. J Coll Inter Sci, 300, 131-140.
- Silverman L, Boskey AL (2004): Diffusion systems for evaluation of biomineralization. Calcif Tissue Int, 75, 494-501.
- Sodek J, Ganss B, Mckee MD (2000): Osteopontin. Crit Rev Oral Biol. Med, 11, 279-303.
- Sun BS, You J, Li Y ve ark. (2013): Osteopontin knockdown suppresses non small cell lung cancer cell invasion and metastasis. Chin Med J, 126, 1683-1688.
- Svennevig K, Prydz K, Kolset SO (1995): Proteoglycans in polarized epithelial Madin-Darbycanine kidney cells. Biochem J, 311, 881-888.
- Taller A, Grohe B, Rogers KA ve ark. (2007): Specific adsorption of osteopontin and synthetic polypeptides to calcium oxalate monohydrate crystals. Biophys J, 93, 1768-1777.
- Taşçene N, İsgüder Z, Salmanoğlu B (2014): Osteopontin expression in polarized MDCK cells. Kafkas Univ Vet Fak Derg, 20, 671-674.
- Ulrich O, Mann K, Haase W ve ark. (1991): Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line. J Biol Chem, 266, 3518-3525.
- Umekawa T, Iguchi M, Uemura H ve ark. (2006): Oxalate ions and calcium oxalate crystal-induced up- regulation of osteopontin and monocyte chemoattractant protein-1 in renal fibroblasts. BJU Int, 98, 656-660.
- Verhulst A, Persy VP, Rompay AV ve ark. (2002): Osteopontin synthesis and localization along the human nephron. J Am Soc Nephrol, 13, 1210-1218.
- Wang L, Guan X, Tang R ve ark. (2008): Phosphorylation of osteopontin is required for inhibition of calcium oxalate crystallization. J Phys Chem B, 112, 9151-9157.
- Wesson JA, Johnson RJ, Mazzali M ve ark. (2003): Osteopontin is a critical inhibitör of calcium oxalate crystal formation and retention in renal tubules. J Am Soc Nephrol, 14, 139-47.
- Xie Y, Sakatsume M, Nishi S ve ark. (2001): Expression, roles, receptors and regulation of osteopontin in the kidney. Kidney Int, 60, 1645-1657.
- Yamaguchi S, Wiessner J, Hasegawa A ve ark. (2002): Calcium oxalate monohydrate crystal binding substance produced from Madin-Darby canine kidney cells. Int J Urol, 9, 50-58.
- Yamate T, Kohri K, Umekawa T ve ark. (1998): Interaction between osteopontin on Madin-Darby canine kidney cell membrane and calcium oxalate crystal. Urol Int, 62, 81-86.
- Yamate T, Tsuji H, Amasaki N ve ark. (2000): Analysis of osteopontin DNA in patients with urolithiasis. Urol Res, 28, 159-166.
- Yasui T, Sato M, Fujıta K ve ark. (2001): Effects of citrate on renal stone formation and osteopontin in a rat urolithiasis model. Urol Res, 29, 50-56. Geliş tarihi: 01.07.2015 / Kabul tarihi: 09.08.2016
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Veteriner Cerrahi |
| Diğer ID | JA64UB63ZM |
| Bölüm | Araştırma Makalesi |
| Yazarlar | |
| Yayımlanma Tarihi | 1 Haziran 2017 |
| Yayımlandığı Sayı | Yıl 2017Cilt: 64 Sayı: 2 |