Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
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Effects of puromycin aminonucleoside on the cytoskeletal changes of glomerular epithelial cells

Puromycin aminonucleoside의 사구체 상피세포에 대한 영향

  • Lee, Jun Ho (Department of Pediatrics, Pochon CHA University, College of Medicine) ;
  • Ha, Tae Sun (Department of Pediatrics, Chungbuk National University, College of Medicine)
  • 이준호 (포천중문의대 분당차병원 소아청소년과) ;
  • 하태선 (충북대학교 의과대학 소아과학교실)
  • Received : 2007.10.11
  • Accepted : 2007.11.25
  • Published : 2008.01.15
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Abstract

Purpose : This study was designed to clarify the mechanism of proteinuria in nephrotic syndrome patients by using puromycin aminonucleoside (PAN) nephrosis model. Methods : Following administration of various concentrations of PAN and antioxidants we observed the changes of podocyte cytoskeletons in cultured rat glomerular epithelial cells (GEpC) by method of scanning electron microscope, reactive oxyten species (ROS) analysis, permeability assay, confocal microscope, and Western blot assay. Results : PAN not only induced the ultrastructural changes of GEpC, such as shortening and fusion of microvilli, but also separated the intercellular gaps and linear ZO-1. PAN induced oxidative stresses in time and dose dependent manners and increases of intercellular permeability in anti-oxidants inhibitable manners. High concentration of PAN induced not only actin polymerization and disorganization, but also the conglomerulation and internal dislocation of ${\alpha}-actinin$ protein. The intensities of fluorescences of ZO-1 protein were diminished and internalized by PAN in a dose-dependent manner, which were inhibited by anti anti-oxidants. Conclusion : PAN induced the changes of podocytes cytoskeleton and junctional barriers by way of increasing ROS in GEpC that resulted in increasing their permeability in a antioxidatn-inhibitable manner. Glomerular hyperpermeability induced by PAN mediateing through oxidative stresses is thought to take part in the mechanism of proteinuria in nephrotic syndrome.

목 적 : 특발성 신증후군의 연구를 위하여 병태생리 및 임상소견과 유사한 실험적 puromycin aminonucleoside(PAN) 신증을 이용하는데 본 연구는 배양한 사구체 상피세포에 대한 PAN의 영향을 통하여 신증후군의 주 원인 병태생리인 단백뇨의 기전을 밝히고자 하였다. 방 법 : 사구체 상피세포를 배양한 후 다양한 농도의 PAN과 항산화제를 투여하여 전자현미경관찰, 반응성 산소종 투과율 변화, confocal microscopy 등을 통하여 족세포성분의 변화를 관찰하였다. 결 과 : 사구체 상피세포의 초고배율소견에서 PAN에 의해 세포간극이 벌어지고 표면의 미세돌기가 단축되는 변화를 볼 수 있었다. 이러한 세포간극의 변화는 세포막부분의 ZO-1에 대한 면역형광검사에서도 확인할 수 있었다. DCF-DA로 측정한 반응성 산소종은 PAN에 의하여 농도에 따라 투여 2시간에 이미 유의한 증가를 보이나, 이러한 변화는 항산화제인 EGCG, probucol, vitamin C에 의해 감소하였다. 또한, 세포단층모델에서 투과율은 PAN에 의하여 농도에 따라 증가하나 항산화제에 의해 증가가 억제되었다. 세포골격구조인 ${\alpha}-actinin$은 사구체 상피세포의 세포질과 바깥 세포막부분으로 actin과 같이 분포하나 고농도의 PAN에 의해 세포질 바깥쪽의 일부분에 집중하는 형상으로 변하였다. 그러나 이러한 변화는 항산화제인 vitamin C의 처치에 의해 예방될 수 있었다. 세극막성분인 ZO-1는 고농도의 PAN에 의해 안쪽으로 이동하고 집중하는 형상으로 변하였으나, vitamin C의 처치에 의해 예방되었다. 이와 함께 ${\alpha}-actinin$과 ZO-1은 PAN에 의해 단백양이 감소하였으나 이는 항산화제에 의해 예방할 수 있었다. 결 론 : PAN은 사구체 상피세포의 반응성 산소종 생성을 증가시키고, 구조성분의 변화를 통하여 형태학적인 변화를 초래하며 이는 투과율의 증가로 나타났다. 이러한 변화들은 항산화제에 의해 어느 정도 억제할 수 있었음으로, PAN은 생체 외 사구체 상피세포에 산화스트레스기전을 통하여 구조적 변화와 이에 따른 단백뇨를 유발시키는 것으로 사료된다.

Keywords

Acknowledgement

Supported by : 한국과학재단

References

  1. Nash MA, Edelmann CM Jr, Burnstein J, Barnett HL. Minimal change nephrotic syndrome, Diffuse mesangial hypercellularity, and focal glomerular sclerosis. Pediatric Kidney Disease. 2nd ed. Boston: Little, Brown and Company, 1992;1267-90
  2. Ryan GB, Karnovsky MJ. An ultrastructural study of the mechanisms of proteinuria in aminonucleoside nephrosis. Kidney Int 1975;8:219-32 https://doi.org/10.1038/ki.1975.105
  3. Caulfield JP, Reid JJ, Farquhar MG. Alterations of the glomerular epithelium in acute aminonucleoside nephrosis: Evidence of formation of occluding junctions and epithelial cell detachment. Lab Invest 1976;34:43-59
  4. Messina A, Davies DJ, Dillane PC, Ryan GB. Glomerular epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis. Am J Pathol 1987;126:220-9
  5. Caufield JP, Farquhar MG. Loss of anionic sites from the glomerular basement membrane in aminonucleoside nephrosis. Lab Invest 1978;39:505-10
  6. Alcom D, Ryan GB. Distribution of anionic groups in the glomerular capillary wall in rat nerphrotoxic nephritis and aminonucleoside nephrosis. Pathology 1981;13:37-50 https://doi.org/10.3109/00313028109086828
  7. Mynderse LA, Hassell JR, Kleinman HK, Martin GR, Martinez-Hernandez A. Loss of heparin sulfate proteoglycan from glomerular basement membrane of nephrotic rats. Lab Invet 1983;48:292-302
  8. Quatacker J. Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis. Virchows Arch 1986;50:237-47 https://doi.org/10.1007/BF02889904
  9. Fishman JA, Karnovsky MJ. Effects of aminonucleoside of puromycin on glomerular epithelial cells in vitro. Am J Pathol 1985;118:398-407
  10. Ricardo SD, Bertram JF, Ryan GB. Reactive oxygen species in aminonucleoside nephrosis: In vitro studies. Kidney Int 1994;45:1057-69 https://doi.org/10.1038/ki.1994.142
  11. Mundel P, Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 2002;13:3005-15 https://doi.org/10.1097/01.ASN.0000039661.06947.FD
  12. Oh J, Reiser J, Mundel P. Dynamic(re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome. Pediatr Nephrol 2004;19:130-7 https://doi.org/10.1007/s00467-003-1367-y
  13. Kerjaschki D. Caught flat-footed: podocyte damage and the molecular bases of focal glomerulosclerosis. J Clin Invest 2001;108:1583-7 https://doi.org/10.1172/JCI200114629
  14. Kreisberg JI, Hoover RL, Karnovsky MJ. Isolation and characterization of rat glomerular epithelial cells in vitro. Kidney Int 1978;14:21-30 https://doi.org/10.1038/ki.1978.86
  15. Ha TS. High glucose and advanced glycosylation endproducts(AGE) modulate CD2AP expression in glomerular epithelial cells. Korean J Nephrol 2005;24:709-17
  16. Hammes M, Singh A. Effect of polycations on permeability of glomerular epithelial cell monolayers to albumin. J Lab Clin Med 1994;123:437-46
  17. Pegoraro AA, Singh AK, Arruda JA, Dunea G, Bakir AA. A simple method to detect an albumin permeability factor in the idiopathic nephrotic syndrome. Kidney Int 2000;58:1342-45 https://doi.org/10.1046/j.1523-1755.2000.00291.x
  18. Song CJ, Ha TS, Lee HS, Yun OJ. Effects of angiotensin II on glomerular epithelial cells on permeability model; Role of oxidative stress. Korean J Nephrol 2004;23:396-404
  19. Ha TS. High glucose and advanced glycosylation endproducts(AGE) modulate $\alpha$-actinin-4 expression in glomerular epithelial cells. Korean J Nephrol 2004;23:694-702
  20. Kurihara H, Anderson JM, Kerjaschki D, Farquhar MG. The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protaimine sulfate-treated rats contain the tight junction protein ZO-1. Am J Pathol 1992;70:1585-90
  21. Groggel GC, Stevenson J, Hovingh P, Linker A, Border WA. Changes in heparan sulfate correlate with increased glomerular permeability. Kidney Int 1988;33:517-23 https://doi.org/10.1038/ki.1988.28
  22. Daniels BS. The role of the glomerular epithelial cell in the maintenance of the glomerular filtration barrier. Am J Nephrol 1993;13:318-23 https://doi.org/10.1159/000168646
  23. Nash MA, Edelmann CM Jr, Burnstein J, Barnett HL. Minimal change nephrotic syndrome, Diffuse mesangial hypercellularity, and focal glomerular sclerosis. Pediatric Kidney Disease. 2nd ed. Boston: Little, Brown and Company, 1992;1267-1290
  24. Walker F. The origin, turnover and removal of glomerular basement membrane. J Pathol 1973;110:233-44 https://doi.org/10.1002/path.1711100306
  25. Farquhar MG. The primary glomerular filtration barrierbasement membrane or epithelial cells? Kidney Int 1975;8:197-211 https://doi.org/10.1038/ki.1975.103
  26. Kerjaschki D. Dysfunctions of cell biological mechanisms of visceral epithelial cell(podocyte) in glomerular diseases. Kidney Int 1994;45:300-13 https://doi.org/10.1038/ki.1994.39
  27. Ryan GB, Karnovsky MJ. An ultrastructural study of the mechanisms of proteinuria in aminonucleoside nephrosis. Kidney Int 1975;8:219-32 https://doi.org/10.1038/ki.1975.105
  28. Caulfield JP, Reid JJ, Farquhar MG. Alterations of the glomerular epithelium in acute aminonucleoside nephrosis: Evidence of formation of occluding junctions and epithelial cell detachment. Lab Invest 1976;34:43-59
  29. Messina A, Davies DJ, Dillane PC, Ryan GB. Glomerular epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis. Am J Pathol 1987;126:220-9
  30. Caufield JP, Farquhar MG. Loss of anionic sites from the glomerular basement membrane in aminonucleoside nephrosis. Lab Invest 1987;39:505-10
  31. Alcorn D, Ryan GB. Distribution of anionic groups in the glomerular capillary wall in rat nephrotoxic nephritis and aminonucleoside nephrosis. Pathology 1981;13:37-50 https://doi.org/10.3109/00313028109086828
  32. Mynderse LA, Hassell JR, Kleinman HK, Martin GR, Martinez-Hernandez A. Loss of heparan sulfate proteoglycan from glomerular basement membrane of nephrotic rats. Lab Invest 1983;48:292-302
  33. Quatacker J. Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis. Virchows Arch 1986;50:237-47 https://doi.org/10.1007/BF02889904
  34. Glasser RJ, Velosa JA, Michel AF. Experimental model of focal sclerosis.(1): Relationship to protein excretion in aminonucleoside nephrosis. Lab Invest 1977;36:519-26
  35. Fishman JA, Karnovsky MJ. Effects of aminonucleoside of puromycin on glomerular epithelial cells in vitro. Am J Pathol 1985;118:398-407
  36. Weinstein T, Cameron R, Katz A, Silverman M. Rat glomerular epithelial cells in culture express characteristics of parietal, not visceral, epithelium. J Am Soc Nephrol 1992;1279-87
  37. Diamond JR, Pesek I. Glomerular tumor necrosis factor and interleukin 1 during acute aminonucleoside nephrosis: An immunohistochemical study. Lab Invest 1991;64:21-7
  38. Gomez-Chiarri M, Ortiz A, Lerma JL, Lopes-Armada JL, Mampaso F, Gonzalez E, et al. Involvement of tumor necrosis factor and platelet-activating factor in the pathogenesis of experimental nephrosis in rats. Lab Invest 1994;70:449-59
  39. Shibouta Y, Terashita Z, Imura Y, Shino A, Kawamura M, Ohtsuki K et al. Involvement of thromboxane A2, leukotrienes and free radicals in puromycin aminonucleoside nephrosis in rats. Kidney Int 1991;39:920-9 https://doi.org/10.1038/ki.1991.115
  40. Shah SV. Role of reactive oxygen metabolites in experimental glomerular disease. Kidney Int 1986;35:1093-106 https://doi.org/10.1038/ki.1989.96
  41. Trachtman H, Del Pizzo R, Futterweit S. Taurine attenuates renal disease in chronic puromycin aminonucleoside nephropathy. Am J Physiol 1992;262:F117-23
  42. Kawaguchi M, Yamada M, Wada H, Okigaki T. Roles of active oxygen species in glomerular epithelial cell injury in vitro caused by puromycin aminonucleoside. Toxicology 1992;72:329-40 https://doi.org/10.1016/0300-483X(92)90183-F
  43. Ricardo SD, Bertram JF, Ryan GB. Antioxidants protect podocyte foot processes in puromycin aminonucleosidetreated rats. J Am Soc Nephrol 1994;4:1974-86
  44. Ricardo SD, Bertram JF, Ryan GB. Reactive oxygen species in aminonucleoside nephrosis: In vitro studies. Kidney Int 1994;45:1057-69 https://doi.org/10.1038/ki.1994.142
  45. Diamond JR, Karnovsky MJ. Exacerbation of chronic aminonucleoside nephrosis by dietary cholesterol. Kidney Int 1987;2:671-7
  46. Fujiwara Y. An ultrastructural study of the effect of the steroid in Puromycin aminonucleoside nephrosis in rats abstract . Virchows Arch 1984;405:11-24 https://doi.org/10.1007/BF00694922
  47. Bertelli R, Ginevri F, Gusmano R, Ghiggeri GM. Cytotoxic effect of adriamycin and agarose-coupled adriamycin on glomerular epithelial cells: Role of free radicals in vitro cell. Dev Biol 1991;27:799-804 https://doi.org/10.1007/BF02631246

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