單側阻塞性尿路疾病是指尿液無法經由腎臟或輸尿管流入膀胱，尿液迴流造成腎水腫。其發生原因如單側輸尿管結石、狹窄、腫瘤、受傷等，但仍以腎結石最常見，而單側輸尿管阻塞則是一種適合模擬阻塞性尿路腎臟漸進式纖維化發展過程的病理模型。本研究主要在探討單側輸尿管阻塞造成腎臟損傷過中，活性氧、細胞凋亡與細胞自噬的角色以及細胞凋亡與細胞自噬的關係。本實驗利用大白屬於其右側輸尿管靠近腎臟三分之ㄧ處用兩條繩子分別打結，接著縫合腹腔。之後將老鼠隨機分成八組，第一組無進行結紮手術，其餘分別於術後4hr、8hr、12hr、1天、3天、5天、7天進行生理實驗並抽取500μl腎靜脈血進行自由基測定，另外動物犧牲後取阻塞腎臟，利用西方墨點法測定蛋白表現量；使用免疫組織染色觀察腎臟組織型態。結果發現腎靜脈血液H2O2/OH.-類的自由基於阻塞後四至十二小時期間有上升趨勢，而Catalase和MnSOD的蛋白表現從阻塞四小時開始就隨時間下降；腎臟組織Bax/Bcl-2比值及Caspase 3蛋白表現表現則增加；自噬作用的起始蛋白Beclin1及中期蛋白Atg5-Atg12和後期蛋白LC3表現則在阻塞3天後開始逐漸增加，從切片染色結果也發現自噬作用發生在腎臟近端與遠端腎小管處。我們的結果發現自噬作用蛋白和凋亡作用蛋白表現時間一致，表示自噬作用在單側輸尿管阻塞動物模式中有協同細胞死亡的功能。

Unilateral obstructive uropathy refers to the structural changes, like ureteral cot, stenosis, tumor and damage, that impede the normal flow of urine and the most common cause is renal calculus. Unilateral ureteral obstruction (UUO) is an animal model of mimic progressive fibrosis renal disease that can reproducibly demonstrate this pathological progress. The aim of the present study was to investigate the role of reactive oxygen species , apoptosis, autophagy and the relation between apoptosis and autophagy in UUO model. In this research, female wistar rats were performed ligation of the right ureter at the ureteropelvic junction using double silk suture. Rats were divided into 8 groups and the first group had no UUO operation and others were sacrificed after operating UUO for 4hrs, 8hrs, 12hrs, 1 day, 3 days, 5 days, 7 days. After then, rats were proceeded with physiological observation, 500μl volume of renal venous blood for free radical detection, harvested obstruction kidney for protein expression with western blot assay and immunohistochemistry stain for morphology changes. Our results found that, renal venous blood had increased hydrogen peroxide/hydroxyl radical production after UUO 4hrs to 12hrs. Catalase and MnSOD protein expression was decreased with a time-dependent manner from obstruction 4hrs. Bax and Bcl-2 ratio and Caspase 3 protein expression of UUO kidney was increased. UUO enhanced autophagy step Ⅰ protein, Beclin-1； step Ⅱ protein, Atg5-Atg12 complex； step Ⅲ protein, LC3 Ⅱ in a time-dependent after 3 days of UUO. We observed an incremental autophagy and apoptosis protein expression in renal proximal and distal tubules by immunohistochemistry. These results suggest that autophagy and apoptosis have identical tendency and represents that autophagy may play a role with apoptotic cell death in UUO animal models.

1. Agarraberes, F.A., Terlecky, S.R., Dice, J.F., 1997. An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation. J. Cell Biol. 137, 825-834.
2. Ahlberg J, M.L., Glaumann H., 1982. Uptake and degradation of proteins by isolated rat liver lysosomes. Suggestion of a microautophagic pathway of proteolysis. Lab Invest 47, 523.
3. Anders, H.-J., Vielhauer, V., Frink, M., Linde, Y., Cohen, C.D., Blattner, S.M., Kretzler, M., Strutz, F., Mack, M., Grone, H.-J., Onuffer, J., Horuk, R., Nelson, P.J., Schlondorff, D., 2002. A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation, pp. 251-259.
4. Barnhart, B.C., Pietras, E.M., Algeciras-Schimnich, A., Salmena, L., Sayama, K., Hakem, R., Peter, M.E., 2004. CD95 Apoptosis resistance in certain cells can be overcome by noncanonical activation of caspase-8. Cell Death Differ 12, 25-37.
5. Bascands, J.L., Schanstra, J.P., 2005. Obstructive nephropathy: insights from genetically engineered animals. Kidney Int 68, 925-937.
6. Blommaart, E.F., Luiken, J.J., Meijer, A.J., 1997. Autophagic proteolysis: control and specificity. Histochem J 29, 365-385.
7. Bredesen, D.E., 2007. Key Note Lecture: Toward a Mechanistic Taxonomy for Cell Death Programs. Stroke 38, 652-660.
8. Chevalier, R.L., 1999. Molecular and cellular pathophysiology of obstructive nephropathy. Pediatr Nephrol 13, 612-619.
9. Chevalier, R.L., Thornhill, B.A., Wolstenholme, J.T., Kim, A., 1999. Unilaeral ureteral obstruction in early development alters renal growth: dependemce on the duration of obstruction. J Urol 161, 309-313.
10. Chiang, H.L., Terlecky, S.R., Plant, C.P., Dice, J.F., 1989. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246, 382-385.
11. Chien, C.T., Lee, P.H., Chen, C.F., Ma, M.C., Lai, M.K., Hsu, S.M., 2001a. De novo demonstration and co-localization of free-Radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion. J Am Soc Nephrol 12, 973-982.
12. Cochrane, A.L., Kett, M.M., Samuel, C.S., Campanale, N.V., Anderson, W.P., Hume, D.A., Little, M.H., Bertram, J.F., Ricardo, S.D., 2005. Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol 16, 3623-3630.
13. Codogno, P., Meijer, A.J., 2005. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 12, 1509-1518.
14. Cuervo, A.M., 2004. Autophagy: many paths to the same end. Mol Cell Biochem 263, 55-72.
15. Dean, R.T., 1977. Lysosomes and membrane recycling. A hypothesis. Biochem. J. 168, 603-600.
16. Docherty, N.G., O'Sullivan, O.E., Healy, D.A., Fitzpatrick, J.M., Watson, R.W., 2006. Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction. Am J Physiol Renal Physiol 290, F4-13.
17. Dunn, W.A., Jr., 1994. Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol 4, 139-143.
18. Eddy, A.A., 1994. Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions. J Am Soc Nephrol 5, 1273-1287.
19. Edinger, A.L., Thompson, C.B., 2004. Death by design: apoptosis, necrosis and autophagy. Curr. Opin. Cell. Biol. 16, 663-669.
20. Englert, R.P., Shacter, E., 2002a. Distinct modes of cell death induced by different reactive oxygen species. J. Biol. Chem. 277, 20518-20526.
21. Englert, R.P., Shacter, E., 2002b. Distinct modes of cell death induced by different reactive oxygen species. Amino acyl chloramines mediate hypochlorous acid-induced apoptosis. J. Biol. Chem. 277, 20518-20526.
22. Esteban, V., Lorenzo, O., Rupe'rez, M., Suzuki, Y., Mezzano, S., Blanco, J., Kretzler, M., Sugaya, T., Egido, J., Ruiz-Ortega, M., 2004. Angiotensin II, via AT1 and AT2 receptors and NF-B Pathway, regulates the inflammatory response in unilateral ureteral obstruction. J Am Soc Nephrol 15, 1514-1529.
23. Ferraro, E., Cecconi, F., 2007. Autophagic and apoptotic response to stress signals in mammalian cells. Arch Biochem Biophys 462, 210-219.
Friedlander, R.M., 2003. Apoptosis and Caspases in Neurodegenerative
Diseases, pp. 1365-1375.
24. Hanada, T., Noda, N.N., Satomi, Y., Ichimura, Y., Fujioka, Y., Takao, T., Inagaki, F., Ohusmi, Y., 2007. The ATG12-ATG5 conjugate has a novel e3-like activity for protein lipidation in autophagy, p. C700195200.
25. Hegarty, N.J., Young, L.S., Kirwan, C.N., O'Neill, A.J., Bouchier-Hayes, D.M., Sweeney, P., Watson, R.W.G., Fitzpatrick, J.M., 2001. Nitric oxide in unilateral ureteral obstruction: Effect on regional renal blood flow. Kidney Int 59, 1059-1065.
26. Hershko, A., Ciechanover, A., 1998. The ubiquitin system, pp. 425-479.
27. Hochberg, D., Johnson, C.W., Chen, J., Cohen, D., Stern, J., Vaughan, E.D., Jr., Poppas, D., Felsen, D., 2000. Interstitial fibrosis of unilateral ureteral obstruction is exacerbated in kidneys of mice lacking the gene for inducible nitric oxide synthase. Lab Invest 80, 1721-1728.
28. Kawada, N., Moriyama, T., Ando, A., Fukunaga, M., Miyata, T., Kurokawa, K., Imai, E., Hori, M., 1999. Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction. Kidney Int 56, 1004-1013.
29. Kehrer, J.P., 1993. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 23, 21-48.
30. Kellner, D., Chen, J., Richardson, I., Seshan, S.V., El Chaar, M., Vaughan, E.D., Jr., Poppas, D., Felsen, D., 2006. Angiotensin receptor blockade decreases fibrosis and fibroblast expression in a rat model of unilateral ureteral obstruction. J Urol 176, 806-812.
31. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239-257.
32. Kipari, T., Cailhier, J.-F., Ferenbach, D., Watson, S., Houlberg, K., Walbaum, D., Clay, S., Savill, J., Hughes, J., 2006. Nitric Oxide Is an Important Mediator of Renal Tubular Epithelial Cell Death in Vitro and in Murine Experimental Hydronephrosis, pp. 388-399.
33. Kirkegaard, K., Taylor, M.P., Jackson, W.T., 2004. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Micro 2, 301-314.
34. Klionsky, D.J., Emr, S.D., 2000. Autophagy as a Regulated Pathway of Cellular Degradation. Science 290, 1717-1721.
35. Krammer, P.H., 2000. CD95's deadly mission in the immune system. Nature 407, 789-795.
36. Kroemer, G., Galluzzi, L., Brenner, C., 2007. Mitochondrial Membrane Permeabilization in Cell Death. Physiol. Rev. 87, 99-163.
37. Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T., Mizushima, N., 2004. The role of autophagy during the early neonatal starvation period. Nature 432, 1032-1036.
38. Levine, B., 2005. Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 120, 159-162.
39. Levine, B., Yuan, J., 2005. Autophagy in cell death: an innocent convict? J. Clin. Invest. 115, 2679-2688.
40. Liang, X.H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., Levine, B., 1999. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672-676.
41. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent, pp. 265-275.
42. Luo, S., Rubinsztein, D.C., 2007. Atg5 and Bcl-2 provide novel insights into the interplay between apoptosis and autophagy. Cell Death Differ 14, 1247-1250.
43. Maiuri, M.C., Zalckvar, E., Kimchi, A., Kroemer, G., 2007. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8, 741-752.
44. Meldrum, K.K., Metcalfe, P., Leslie, J.A., Misseri, R., Hile, K.L., Meldrum, D.R., 2006. TNF-alpha neutralization decreases nuclear factor-kappaB activation and apoptosis during renal obstruction. J Surg Res 131, 182-188.
45. Miyajima, A., Chen, J., Lawrence, C., Ledbetter, S., Soslow, R.A., Stern, J., Jha, S., Pigato, J., Lemer, M.L., Poppas, D.P., Vaughan, E.D., Felsen, D., 2000. Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int 58, 2301-2313.
46. Miyajima, A., Chen, J., Poppas, D.P., Vaughan, E.D., Jr., Felsen, D., 2001. Role of nitric oxide in renal tubular apoptosis of unilateral ureteral obstruction. Kidney Int 59, 1290-1303.
47. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y., 2004. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15, 1101-1111.
48. Moody, T.E., Vaughn, E.D., Jr., Gillenwater, J.Y., 1975. Relationship between renal blood flow and ureteral pressure during 18 hours of total unilateral uretheral occlusion. Implications for changing sites of increased renal resistance. Invest Urol 13, 246-251.
49. Moretti, L., Cha, Y.I., Niermann, K.J., Lu, B., 2007. Switch between apoptosis and autophagy: radiation-induced endoplasmic reticulum stress? Cell Cycle 6, 793-798.
50. Morrissey, J.J., Ishidoya, S., McCracken, R., Klahr, S., 1996. Nitric oxide generation ameliorates the tubulointerstitial fibrosis of obstructive nephropathy, pp. 2202-2212.
51. Mortimore, G.E., Poso, A.R., 1987. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu. Rev. Nutr. 7, 539-568.
52. Mortimore, G.E., Schworer, C.M., 1977. Induction of autophagy by amino-acid deprivation in perfused rat liver. Nature 270, 174-176.
53. Nagatoya, K., Moriyama, T., Kawada, N., Takeji, M., Oseto, S., Murozono, T., Ando, A., Imai, E., Hori, M., 2002. Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int 61, 1684-1695.
54. Ophascharoensuk, V., Giachelli, C.M., Gordon, K., Hughes, J., Pichler, R., Brown, P., Liaw, L., Schmidt, R., Shankland, S.J., Alpers, C.E., Couser, W.G., Johnson, R.J., 1999. Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis. Kidney Int 56, 571-580.
55. Pat, B., Yang, T., Kong, C., Watters, D., Johnson, D.W., Gobe, G., 2005. Activation of ERK in renal fibrosis after unilateral ureteral obstruction: Modulation by antioxidants. Kidney Int 67, 931-943.
56. Pattingre, S., Tassa, A., Qu, X., Garuti, R., Liang, X.H., Mizushima, N., Packer, M., Schneider, M.D., Levine, B., 2005. Bcl-2 Antiapoptotic Proteins Inhibit Beclin 1-Dependent Autophagy. Cell 122, 927-939.
57. Pimentel, J.L., Jr., Martinez-Maldonado, M., Wilcox, J.N., Wang, S., Luo, C., 1993. Regulation of renin-angiotensin system in unilateral ureteral obstruction. Kidney Int 44, 390-400.
58. Pyo, J.O., Jang, M.H., Kwon, Y.K., Lee, H.J., Jun, J.I., Woo, H.N., Cho, D.H., Choi, B., Lee, H., Kim, J.H., Mizushima, N., Oshumi, Y., Jung, Y.K., 2005. Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death. J Biol Chem 280, 20722-20729.
59. Roth, K.S., Koo, H.P., Spottswood, S.E., Chan, J.C., 2002. Obstructive Uropathy: An Important Cause of Chronic Renal Failure in Children. Clin Pediatr 41, 309-314.
60. Sagai, M., Saito, H., Ichinose, T., Kodama, M., Mori, Y., 1993. Biological effects of diesel exhaust particles. I. In vitro production of superoxide and in vivo toxicity in mouse. Free Radic Biol Med 14, 37-47.
61. Schreiner, G.F., Harris, K.P., Purkerson, M.L., Klahr, S., 1988. Immunological aspects of acute ureteral obstruction: immune cell infiltrate in the kidney. Kidney Int 34, 487-493.
62. Schulsinger, D.A., Gulmi, F.A., Chou, S.Y., Mooppan, U.M., Kim, H., 1997. Activation of endothelium-derived relaxing factor system in acute unilateral ureteral obstruction. J Urol 157, 1951-1956.
63. Shintani, T., Klionsky, D.J., 2004. Autophagy in health and disease: a double-edged sword. Science 306, 990-995.
64. Steinman, R.M., Mellman, I.S., Muller, W.A., Cohn, Z.A., 1983. Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96, 1-27.
65. Sugiyama, H., Kobayashi, M., Wang, D.-H., Sunami, R., Maeshima, Y., Yamasaki, Y., Masuoka, N., Kira, S., Makino, H., 2005. Telmisartan inhibits both oxidative stress and renal fibrosis after unilateral ureteral obstruction in acatalasemic mice, pp. 2670-2680.
66. Taneda, S., Hudkins, K.L., Topouzis, S., Gilbertson, D.G., Ophascharoensuk, V., Truong, L., Johnson, R.J., Alpers, C.E., 2003. Obstructive Uropathy in Mice and Humans: Potential Role for PDGF-D in the Progression of Tubulointerstitial Injury, pp. 2544-2555.
67. Tanida, I., Ueno, T., Kominami, E., 2004. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36, 2503-2518.
68. Thorburn, A., 2007. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis.
69. Vaux, D.L., Korsmeyer, S.J., 1999. Cell Death in Development. Cell 96, 245-254.
70. Wang, C.W., Klionsky, D.J., 2003. The molecular mechanism of autophagy. Mol Med 9, 65-76.
71. Wen, J.G., Frokiaer, J., Jorgensen, T.M., Djurhuus, J.C., 1999. Obstructive nephropathy: an update of the experimental research. Urol Res 27, 29-39.
72. Yan, Z.C., Chen, D., Wu, X.Z., Xie, G.R., Ba, Y., Yan, Z., 2007. Effects of aqueous extracts of Aconitum carmichaeli, Rhizoma bolbostemmatis, Phytolacca acinosa, Panax notoginseng and Gekko swinhonis Guenther on Bel-7402 cells. World J Gastroenterol 13, 2743-2746.
73. Yarger, W.E., Schocken, D.D., Harris, R.H., 1980. Obstructive nephropathy in the rat: possible roles for the renin-angiotensin system, prostaglandins, and thromboxanes in postobstructive renal function. J Clin Invest 65, 400-412.
74. Yousefi, S., Perozzo, R., Schmid, I., Ziemiecki, A., Schaffner, T., Scapozza, L., Brunner, T., Simon, H.-U., 2006. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 8, 1124-1132.
75. Zhu, W., Cowie, A., Wasfy, G.W., Penn, L.Z., Leber, B., Andrews, D.W., 1996. Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types. EMBO J 15, 4130-4141.