臺灣博碩士論文加值系統

中文摘要
【目的】
脂肪肝(hepatic steatosis)是全球流行最廣的慢性肝病。近年來，國人在末期腎病之發生率和盛行率皆名列前茅，盛行率仍高居世界第一。本次實驗旨在研究脂肪肝動物模式對腎臟損傷之影響，並觀察TNF-α在其中所扮演的病生理角色。
【方法】
本實驗先用高脂飼料(high fat diet)來誘發C57BL/6J 公鼠(male mice) 產生脂肪肝。然後，分組用etanercept (TNF-α blockade) 或vehicle注射一個月。注射完，測量TNF-α，total cholesterol (TC)， triglyceride (TG)，LDL-cholesterol (LDL-C) 及HDL-cholesterol (HDL-C)。腎臟損傷由組織切片及測量血清 BUN and creatinine來觀察。並且，使用Western blot來測量C57BL/6J 公鼠的腎臟組織及培養的腎隔細胞(renal mesangial cell)中Peroxisome proliferator-activated receptors-delta (PPAR-delta)的表現。
【結果】
血清TNF-α濃度在產生脂肪肝的C57BL/6J 公鼠有顯著的升高，而etanercept改善了脂肪肝，但並未影響TC，TG， HDL-C， LDL-C 及 TNF-α濃度。etanercept顯示其腎臟保護功能因觀察到腎功能改善(recovery of serum BUN and creatinine levels)及腎組織損傷減少。腎組織PPAR-delta的表現也藉由etanercept之注射得著改善，TNF-α 對PPAR-delta 表現之影響亦在培養的腎隔細胞直接觀察到。
【結論】
本次實驗先用餵食高脂飼料誘發C57BL/6J 公鼠產生脂肪肝，發現所引起的腎臟損傷是由TNF-α上升並引發腎臟PPAR-delta 下降而產生，etanercept可以抑制TNF-α的作用，進而改善上述實驗中C57BL/6J 公鼠的腎功能及PPAR-delta之表現。

Abstract
Aim: The present study is designed to investigate the role of TNF-α in renal damage observed in mice with hepatic steatosis.
Materials and Methods: We induced hepatic steatosis in mice using high fat diet and treated mice with etanercept at the dose sufficient to block TNF-α receptors or vehicle for 1 month. Plasma TNF-α, total cholesterol (TC), triglyceride (TG), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) were determined at the end of this treatment. Renal damage was identified by histologic observation and the higher of serum BUN and creatinine. Also, changes of PPAR-delta in kidney and renal mesangial cell (RMC) were analyzed using Western blot.
Results: Serum TNF-α was markedly raised in mice showing hepatic steatosis. However, the levels of blood lipids (TC, TG, HDL-C and LDL-C) and TNF-α were not modified by the treatment of etanercept although the hepatic steatosis has been improved. Etanercept shows renal protection from histological identification and recovery of serum BUN and creatinine levels. Moreover, restoration of PPAR-delta expression by etanercept was observed in mice kidney. Direct effect of TNF-α on PPAR-delta expression was also characterized in RMC cell.
Conclusion: The present study suggested that renal damage in mice with hepatic steatosis is mainly induced by increase of TNF-α through the decrease of renal PPAR-delta. Etanercept could block TNF-α to restore PPAR-delta and improve renal function in mice with hepatic steatosis.

目錄 Ⅰ
英文縮寫 Ⅲ
中文摘要 Ⅳ
英文摘要 Ⅵ
壹、導論 1
一、慢性腎衰竭 1
二、脂肪肝 2
三、慢性腎衰竭和肥胖及脂肪肝的關係 3
1. 慢性腎衰竭和肥胖的關係 3
2. 慢性腎衰竭和脂肪肝的關係 3

四、Peroxisome proliferator-activated receptors (PPARs). 5
1. Peroxisome. 5
2. Peroxisome proliferator-activated receptors 5

五、TNF-α 7
1. TNF-α. 7
2. TNF-α blocker: etanercept 8

貳、研究構想 9
參、研究材料與方法 10
一、實驗藥品 10
二、實驗動物 10
三、實驗步驟 10
1. 動物實驗 10
2. 測量TNF-α level. 11
3. 組織切片. 11
4. 測量活性氧自由基(reactive oxygen species) 12
5. 西方墨點法分析(Western blotting analysis). 12
6. 細胞培養(Cell cultures) 13
7. 統計分析 14

肆、結果 14
ㄧ、TNF-α blockade (etanercept )對血脂及肝臟的影響 14
二、TNF-α blockade (etanercept )對腎臟功能及腎組織切片的影響 15
三、TNF-α 的表現 15
四、PPAR-? 在高脂飼料公鼠腎臟的表現 15
五、活性氧自由基(reactive oxygen species; ROS)的變化 16
伍、討論 17
陸、結論 20
柒、參考文獻 21
捌、圖表 32

References
1. USRD 2009 ADR/reference tables. Available: http://www.usrds.org/2011/view/v2_12.asp
2. NKF KDOQI GUIDELINES Available: http://www.kidney.org/professionals/KDOQI/guidelines_ckd/

3. Fan JG, Jia JD, Li YM, Wang BY, Lu LG, Shi JP, Chan LY; Guidelines for the diagnosis and management of nonalcoholic fatty liver disease: update 2010. Journal of Digestive Diseases 2011; 12:38-44.
4.Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 2003; 37: 1202-1219
5.Machado M, Marques-Vidal P, Cortez-Pinto H. Hepatic histology in obese patients undergoing bariatric surgery. Journal of Hepatology 2006; 45: 600-606
6.Sanyal AJ. AGA Technical review on nonalcoholic fatty liver disease. Gastroenterology 2002; 123: 1705-1725
7.Lewis JR, Mohanty SR. Nonalcoholic fatty liver disease: a review and update. Digestive Diseases and Sciences 2010; 55: 560-578
8. Tung TH, Chang TH, Chiu WH, Lin TH, Shih HC, Chang MH, Liu JH. Clinical correlation of nonalcoholic fatty liver disease in a Chinese taxi drivers population in Taiwan: Experience at a teaching hospital. BMC Research Notes. 2011; 4:315.
9. Coresh J, Astor BC, Greene T, Eknoyan G, Levey AS. Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey. American Journal of Kidney Disease 2003; 41:1-12.
10.Kalaitzidis RG, Siamopoulos KC. The role of obesity in kidney disease: recent findings and potential mechanisms. International Urology and Nephrology 2011; 43: 771-784
11.Yun KE, Shin CY, Yoon YS, Park HS. Elevated alanine aminotransferase levels predict mortality from cardiovascular disease and diabetes in Koreans. Atherosclerosis 2009; 205: 533-537
12.Targher G, Kendrick J, Smits G, Chonchol M. Relationship between serum gamma-glutamyltransferase and chronic kidney disease in the United States adult population. Findings from the National Health and Nutrition Examination Survey 2001-2006. Nutrition, Metabolism, and Cardiovascular Diseases (NMCD) 2010; 20: 583-590
13.Targher G, Bosworth C, Kendrick J, Smits G, Lippi G, Chonchol M. Relationship of serum bilirubin concentrations to kidney function and albuminuria in the United States adult population. Findings from the National Health and Nutrition Examination Survey 2001-2006. Clinical Chemistry and Laboratory Medicine (CCLM / FESCC) 2009; 47: 1055-1062
14.Targher G, Bertolini L, Chonchol M, Rodella S, Zoppini G, Lippi G, Zenari L, Bonora E. Non-alcoholic fatty liver disease is independently associated with an increased prevalence of chronic kidney disease and retinopathy in type 1 diabetic patients. Diabetologia 2010; 53: 1341-1348
15.Targher G, Bertolini L, Rodella S, Zoppini G, Lippi G, Day C, Muggeo M. Non-alcoholic fatty liver disease is independently associated with an increased prevalence of chronic kidney disease and proliferative/laser-treated retinopathy in type 2 diabetic patients. Diabetologia 2008; 51: 444-450
16.Yilmaz Y, Alahdab YO, Ozdogan O, Dolar E. Non-alcoholic fatty liver disease and microalbuminuria in non-diabetic patients: role of insulin resistance. Internal Medicine Journal 2009; 39: 709-710
17.Targher G, Bertolini L, Rodella S, Lippi G, Zoppini G, Chonchol M. Relationship between kidney function and liver histology in subjects with nonalcoholic steatohepatitis. Clinical Journal of the American Society of Nephrology (CJASN) 2010; 5: 2166-2171
18.Machado MV, Goncalves S, Carepa F, Coutinho J, Costa A, Cortez-Pinto H. Impaired renal function in morbid obese patients with nonalcoholic fatty liver disease. Liver International : Official Journal of the International Association for the Study of the Liver 2012; 32: 241-248
19. Gabald?n T . Peroxisome diversity and evolution. Philosophical Transactions of the Royal Society of London. Series B, Biologicl Sciences 2010; 365: 765–773
20. Wanders RJ, Waterham HR. Biochemistry of mammalian peroxisomes revisited. Annual Review of Biochemistry 2006; 75: 295–332
21. Berger J, G?rtner J. X-linked adrenoleukodystrophy: clinical, biochemical and pathogenetic aspects. Biochimica et Biophysica Acta. 2006; 1763: 1721-1732.
22.Schupp M, Lazar MA. Endogenous ligands for nuclear receptors: digging deeper. The Journal of Biological Chemistry 2010; 285: 40409-40415
23.Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 1999; 20: 649-688
24.Cowherd RM, Lyle RE, McGehee RE, Jr. Molecular regulation of adipocyte differentiation. Seminars in Cell & Developmental Biology 1999; 10: 3-10
25.Staels B, Maes M, Zambon A. Fibrates and future PPARalpha agonists in the treatment of cardiovascular disease. Nature Clinical Practice Cardiovascular Medicine 2008; 5: 542-553
26.Zandbergen F, Plutzky J. PPARalpha in atherosclerosis and inflammation. Biochimica et Biophysica Acta 2007; 1771: 972-982
27.Adkins Y, Kelley DS. Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids. The Journal of Nutritional Biochemistry 2010; 21: 781-792
28.Rosenson RS. Fenofibrate: treatment of hyperlipidemia and beyond. Expert Review of Cardiovascular Therapy 2008; 6: 1319-1330
29.Glass CK, Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells. Nature Reviews Immunology 2010; 10: 365-376
30.Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, Morel CR, Subramanian V, Mukundan L, Red Eagle A, Vats D, Brombacher F, Ferrante AW, Chawla A. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature 2007; 447: 1116-1120
31.Moller DE, Berger JP. Role of PPARs in the regulation of obesity-related insulin sensitivity and inflammation. International journal of obesity and related metabolic disorders : Journal of the International Association for the Study of Obesity 2003; 27 Suppl 3: S17-21
32.Yang T, Michele DE, Park J, Smart AM, Lin Z, Brosius FC, 3rd, Schnermann JB, Briggs JP. Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney. The American Journal of Physiology 1999; 277: F966-973
33.Collino M, Benetti E, Miglio G, Castiglia S, Rosa AC, Aragno M, Thiemermann C, Fantozzi R. Peroxisome proliferator-activated receptor beta/delta agonism protects the kidney against ischemia/reperfusion injury in diabetic rats. Free Radical Biology & Medicine 2011; 50: 345-353
34. Kolb WP, Granger GA. Lymphocyte in vitro cytotoxicity: characterization of human lymphotoxin. Proceeding of National Academy of Science of the United States of America. 1968; 61: 1250–1255.
35. Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, Palladino MA, Kohr WJ, Aggarwal BB, Goeddel DV. Tumor necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 1984; 312: 724– 729.
36. Beutler B, Milsark IW, Cerami AC. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science 1985; 229: 869–871.
37. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 2001; 104: 487–501.
38.Uysal S, Armutcu F, Aydogan T, Akin K, Ikizek M, Yigitoglu MR. Some inflammatory cytokine levels, iron metabolism and oxidan stress markers in subjects with nonalcoholic steatohepatitis. Clinical Biochemistry 2011; 44: 1375-1379
39.Lee TI, Kao YH, Chen YC, Pan NH, Chen YJ. Oxidative stress and inflammation modulate peroxisome proliferator-activated receptors with regional discrepancy in diabetic heart. European Journal of Clinical Investigation 2010; 40: 692-699
40. Karsten Peppel, David Crawford, and Bruce Beutler. A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity. The Journal of Experimental Medicine 1991; 174: 1483-1489
41. Braun J, McHugh N, Singh A, Wajdula JS, Sato R. Improvement in patient-reported outcomes for patients with ankylosing spondylitis treated with etanercept 50?mg once-weekly and 25?mg twice-weekly. Rheumatology (Oxford) 2007; 46: 999–1004.
42. FDA Available: http://www.fda.gov/bbs/topics/ANSWERS/2003/ANS01240.html
43. Pang M, de la Monte SM, Longato L, Tong M, He J, Chaudhry R, Duan K, Ouh J, Wands JR. PPARdelta agonist attenuates alcohol-induced hepatic insulin resistance and improves liver injury and repair. Journal of Hepatology 2009; 50: 1192-1201
44. Song HL, Lv S, Liu P. The roles of tumor necrosis factor-alpha in
colon tight junction protein expression and intestinal mucosa
structure in a mouse model of acute liver failure. BMC Gastroenterology 2009; 9: 70. doi: 10.1186/1471-230X-9-70.
45.Hsieh TJ, Zhang SL, Filep JG, Tang SS, Ingelfinger JR, Chan JS. High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells. Endocrinology 2002; 143: 2975-2985
46.Xiong Z, Huang H, Li J, Guan Y, Wang H. Anti-inflammatory effect of PPARgamma in cultured human mesangial cells. Renal Failure. 2004; 26:497-505.
47. Zu N, Li P, Li N, Choy P, Gong Y. Mechanism of saikosaponin-d in the regulation of rat mesangial cell proliferation and synthesis of extracellular matrix proteins. Biochemistry and Cell Biology.2007 85:169-74.
48. Eccleston HB, Andringa KK, Betancourt AM, King AL, Mantena SK, Swain TM, Tinsley HN, Nolte RN, Nagy TR, Abrams GA, Bailey SM. Chronic exposure to a high-fat diet induces hepatic steatosis, impairs nitric oxide bioavailability, and modifies the mitochondrial proteome in mice. Antioxidants & Redox Signaling 2011; 15: 447-459
49.Mauer SM, Steffes MW, Brown DM. The kidney in diabetes. The American Journal of Medicine 1981; 70: 603-612
50. Pfeffer K: Biological functions of tumor necrosis factor cytokines and their receptors. Cytokine Growth Factor Reviews 2003;14: 185-191.
51.Silva LC, Ortigosa LC, Benard G. Anti-TNF-alpha agents in the treatment of immune-mediated inflammatory diseases: mechanisms of action and pitfalls. Immunotherapy 2010; 2: 817-833
52.Liang YJ, Jian JH, Liu YC, Juang SJ, Shyu KG, Lai LP, Wang BW, Leu JG. Advanced glycation end products-induced apoptosis attenuated by PPARdelta activation and epigallocatechin gallate through NF-kappaB pathway in human embryonic kidney cells and human mesangial cells. Diabetes/Metabolism Research and Reviews 2010; 26: 406-416
53.Reyes-Martin P, Alique M, Parra T, Hornedo JP, Lucio-Cazana J. Cyclooxygenase-independent inhibition of H2O2-induced cell death by S-ketoprofen in renal cells. Pharmacological Research (The Official Journal of the Italian Pharmacological Society) 2007; 55: 295-302
54. Neeraja Kambham, Glen S Markowitz, Anthony M Valeri, Julie Lin and Vivette D D''Agati Obesity-related glomerulopathy: an emerging epidemic. Kidney International 2001; 59:1498–1509
55. Askling J, Fahrbach K, Nordstrom B, Ross S, Schmid CH, Symmons D. Cancer risk with tumor necrosis factor alpha (TNF) inhibitors: meta-analysis of randomized controlled trials of adalimumab, etanercept, and infliximab using patient level data. Pharmacoepidemiology and Drug Safety 2011; 20:119-130.
56. Nishanthi Thalayasingam, John D. Isaacs. Anti-TNF therapy. Best Practice & Research Clinical Rheumatology 2011; 25: 549–567