Puromycin aminonucleoside 투여에 따른 사구체 족세포 β-catenin의 변화
The Change of Podocyte β-Catenin by Puromycin Aminonucleoside
2
조회
0
다운로드
3
인용
0
참고문헌
키워드
초록
목적:단백뇨 질환의 실험모델인 puromycin aminonucleoside (PAN)에서 관찰할 수 있는 족세포의 병리학적 이상에 있어서 β-catenin의 변화를 생체 외 족세포 배양실험을 통하여 알아보고자 하였다. 방 법:PAN에 의한 족세포의 β-catenin의 변화를 생체 외 배양실험을 통해 알아보고자 백서 사구체 상피세포를 배양하여 다양한 농도의 PAN을 투여하여 confocal 현미경을 통하여 β-catenin의 분포를 관찰하였고, Western blotting과 RT-PCR을 사용하여 β-catenin 발현의 변화를 관찰하였다. 결과:외곽세포질에 분포하는 β-catenin이 단일세포 혹은 응집환경에서 PAN의 농도가 올라갈수록 흐려지면서 세포간에 간극이 생기는 것을 볼 수 있었다. Western 분석에서, β-catenin 단백양은 PAN의 농도가 증가할수록, 특히, 고농도인 50 μg/ mL에서 24시간과 48시간이 노출 조건에서 각각 34.9%와 34.3%의 의미 있는 감소소견을 보였다 (P<0.05). 이러한 소견은 RT-PCR에서도 유사하게 보였으며, 24시간에서는 고농도인 50 μg/mL PAN을 첨가한 조건에서 25.4%의 의미 있는 감소를 보였으며, 48시간에서는 25 μg/mL와 50 μg/ mL PAN을 첨가한 조건에서 각각 46.6%와 51.8%의 의미 있는 감소를 보였다. 결 론:PAN은 족세포에서 β-catenin을 세포막으로부터 내부로의 분포변화를 유발하고, β-catenin mRNA의 발현 감소와 단백수준에서 양의 감소를 초래함으로서, PAN에 의한 족세포 내 분포변화에 유전자 억제에 의한 β-catenin 단백의 감소로 단백뇨의 발생에 기여할 것이라 사료된다.
Abstract
Purpose:To test whether the expression of β-catenin, a component of podocyte as a filtration molecule, would be altered by puromycin aminonucleoside (PAN) in the cultured podocyte in vitro. Methods:We cultured rat glomerular epithelial cells (GEpC) with various concentrations of PAN and examined the distribution of β-catenin by confocal microscope and measured the change of β-catenin expression by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Results:We found that β-catenin relocalized from peripheral cytoplasm to inner cytoplasm, therefore, intercellular separations were seen in confluently cultured cells by high concentrations of PAN in immunofluorescence views. In Western blotting of GEpC, PAN (50 μg/mL) decreased β-catenin expression by 34.9% at 24 hrs and 34.3% at 48 hrs, compared to those in without PAN condition (P<0.05). In RT-PCR, high concentrations (50 μg/mL) of PAN also decreased β-catenin mRNA expression similar to protein suppression by 25.4% at 24 hrs and 51.8% at 48 hrs (P<0.05). Conclusion:Exposure of podocytes to PAN in vitro relocates β-catenin internally and reduces β-catenin mRNA and protein expression, which could explain the development of proteinuria in experimental PAN-induced nephropathy.
참고문헌 (28)
- Churg J, "Pathology of the nephrotic syndrome in children: a report for the International Study of Kidney Disease in Children", Lancet, 760, pp.1299~1302 (1970)
- Eddy AA, "Nephrotic syndrome in childhood", Lancet, 362, pp.629~639 (2003)
- Ryan GB, "An ultrastructural study of the mechanisms of proteinuria in aminonucleoside nephrosis", Kidney Int, 8, pp.219~232 (1975)
- Caulfield JP, "Alterations of the glomerular epithelium in acute aminonucleoside nephrosis: Evidence of formation of occluding junctions and epithelial cell detachment", Lab Invest, 34, pp.43~59 (1976)
- Messina A, "Glomerular epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis", Am J Pathol, 126, pp.220~229 (1987)
- Caufield JP, "Loss of anionic sites from the glomerular basement membrane in aminonucleoside nephrosis", Lab Invest, 39, pp.505~512 (1978)
- Fishman JA, "Effects of aminonucleoside of puromycin on glomerular epithelial cells in vitro", Am J Pathol, 118, pp.398~407 (1985)
- Ricardo SD, "Reactive oxygen species in aminonucleoside nephrosis:In vitro studies", Kidney Int, 45, pp.1057~1069 (1994)
- Marshall CB, "Puromycin aminonucleoside induces oxidant-dependent DNA damage in podocytes in vitro and in vivo", Kidney Int, 70, pp.1962~1973 (2006)
- Karnovsky MJ, "Ainsworth SK. The structural basis of glomerular filtration", Adv Nephrol Necker Hosp, 2, pp.35~60 (1972)
- Remuzzi A, "Glomerular permselective function", Kidney Int, 45, pp.398~402 (1994)
- Rodewald R, "Porous substructure of the glomerular silt diaphragm in the rat and mouse", J Cell Biol, 60, pp.423~433 (1974)
- Reiser J, "The glomerular silt diaphragm is a modified adherens junction", J Am Soc Nephrol, 11, pp.1~8 (2000)
- Mundel P, "Podocyte biology and response to injury", J Am Soc Nephrol, 13, pp.3005~3015 (2002)
- Haraldsson B, "Properties of the glomerular barrier and mechanisms of proteinuria", Physiol Rev, 88, pp.451~487 (2008)
- Fodde R, "Wnt", b-catenin signaling in cancer stemness and malignant behavior, 2007, pp.19, 150~158
- Arce L, "Diversity of LEF", TCF action in development and disease, 2006, pp.25, 7492~7504
- Khoshnoodi J, "Unraveling the molecular make-Up of the glomerular podocyte slit diaphragm", Exp Nephrol, 9, pp.355~359 (2001)
- Kreisberg JI, "Isolation and characterization of rat glomerular epithelial cells in vitro", Kidney Int, 14, pp.21~30 (1978)
- 하태선, "Effects of advanced glycosylation endproducts on perlecan core protein of glomerular epithelium", PEDIATRIC NEPHROLOGY, 19, pp.1219~1224, SPRINGER
- Yaoita E, "Cadherin and catenin staining in podocytes in development and puromycin aminonucleoside nephrosis", Nephrol Dial Transplant, 17, pp.16~19 (2002)
- Luimula P, "Podocyte-associated molecules in puromycin aminonucleoside nephrosis of the rat", Lab Invest, 82, pp.713~718 (2002)
- Dai C, "Wnt", β-catenin signaling promotes podocyte dysfunction and albuminuria, 2009, pp.20, 1997~2008
- He W, "Blockade of Wnt", β-catenin signaling by paricalcitol ameliorates proteinuria and kidney injury, 2011, pp.22, 90~103
- Heikkilä E, "β-Catenin mediates adriamycin-induced albuminuria and podocyte injury in the adult mouse kidneys", Nephrol Dial Transplant, 25, pp.2437~2446 (2010)
- Lin KH, "Advanced glycation end-productinhibited cell proliferation and protein expression of β-catenin and cyclin D1 are dependent on glycogen synthase kinase 3β in LLC-PK1 cells", Arch Biochem Biophys, 477, pp.27~32 (2008)
- Naves MA, "Podocyte Wnt", ß-catenin pathway is activated by integrin-linked kinase in clinical and experimental focal segmental glomerulosclerosis, 2011, ~
- Heikkilä E, "Densin and betacatenin form a complex and co-localize in cultured podocyte cell junctions", Mol Cell Biochem, 305, pp.9~18 (2007)