Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents ROLE OF INTERLEUKIN-13 ON THE DEVELOPMENT OF MINIMAL CHANGE NEPHROTIC SYNDROME – A NOVEL ANIMAL MODEL LAI KIN WAI (BSc, MMedSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2006 i Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents This thesis is dedicated to Ivy and Joseph that my life will not be complete without them ii Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents ACKNOWLEDGEMENTS I would like to express my thankfulness to: My supervisor, Professor Yap Hui Kim, for her guidance throughout these years. Professor Gilbert Chiang who has always been supportive in my pursuit of a PhD. Mr Larry Poh and Dr Liew Lip Nyin for their valuable advices and from whom I learnt my molecular biology techniques. Dr Cheung Wai, Dr Gong Wei Kin and Dr Tan Puay Hoon for their helpfulness and support. All the friendly and nice people from Department of Paediatrics and other departments who have helped me in a lot of ways on my experiments. This work was supported by grants NMRC 0830/2004 from the National Medical Research Council, Singapore, A*STAR: 04/1/21/19/341 from the Biomedical Research Council, and R-178-000-081-112 from the University Academic Research Fund. i Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents TABLE OF CONTENTS TITLE PAGE DEDICATION ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY ix LIST OF ABBREVIATIONS xi UNITS OF MEASUREMENT xvi LIST OF TABLES xix LIST OF FIGURES xxi LIST OF APPENDICES xxiv LIST OF PUBLICATIONS AND CONFERENCE ABSTRACTS xxvi CHAPTER 1.1 INTRODUCTION Interleukin-13 (IL-13) 1.1.1 Source of IL-13 1.1.2 Gene and protein structure of IL-13 1.1.3 IL-13 receptors 1.1.4 IL-13 and the immune system 1.1.5 Pathological role of IL-13 IL-13, asthma and allergic diseases IL-13 and resistance to gastrointestinal nematodes IL-13 and tissue fibrosis IL-13 and tumor immunosurveillance IL-13 and Hodgkin lymphoma ii Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 1.2 1.3 1.1.6 Transgenic model of IL-13 1.1.7 In-vivo administration of recombinant IL-13 1.1.8 Nephrotic syndrome 10 1.2.1 10 1.5 Definition of nephrotic syndrome 1.2.2 Classification and etiology of nephrotic syndrome 10 Pathogenesis of minimal change nephrotic syndrome 16 1.3.1 Minimal change nephrotic syndrome and the immune system 16 1.3.2 Circulating factors 16 1.3.3 1.4 Knockout model of IL-13 Circulating factors and MCNS 17 Circulating factors and FSGS 19 Bias to Th2 cytokines and involvement of IL-13 20 Genetic aspects of nephrotic syndrome 23 1.4.1 Hereditary diseases manifested with nephrotic syndrome 23 1.4.2 Nephrin and congenital nephrotic syndrome 23 1.4.3 24 Podocin and steroid-resistant nephrotic syndrome 1.4.4 α-Actinin-4 and familial FSGS 25 1.4.5 25 Animal models of nephrotic syndrome Podocyte 27 1.5.1 Podocyte and the glomerular filtration barrier 27 1.5.2 Charge and size selectivity of the glomerular filtration barrier 30 1.5.3 Molecular structure of podocyte foot processes 30 1.5.4 Slit diaphragm 33 1.5.5 The podocyte in idiopathic nephrotic syndrome 34 1.5.6 Mechanism of proteinuria 37 iii Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 1.5.7 Podocyte specific/associated protein molecules implicated in nephrotic syndrome 1.6 Nephrin 41 Podocin 42 Dystroglycan 43 α-Actinin-4 44 CD2AP 45 NEPH1 46 FAT 46 1.5.8 Podocyte and IL-13 47 1.5.9 48 Podocyte and B7-1 Research hypothesis and scope of thesis CHAPTER 2.1 41 MATERIALS & METHODS 49 51 Cloning of rat IL-13 gene into mammalian expression vector 51 2.1.1 51 The pCI mammalian expression vector 2.1.2 Design of primers 53 2.1.3 RT-PCR using Taq polymerase 53 2.1.4 Restriction enzyme digestion of the XhoI and NotI sites 54 2.1.5 Agarose gel electrophoresis and gel extraction of DNA 55 2.1.6 57 Ligation of the rat IL-13 gene and the pCI mammalian expression vector 2.1.7 Transformation of ligation product into E. coli 57 2.1.8 Extraction and purification of plasmid DNA 58 2.1.9 Restriction enzyme digestion reaction (for validation) 58 iv Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 2.1.10 DNA sequencing (for validation) 59 2.2 Large scale production of cloned plasmid 60 2.3 Animal experiments 61 2.3.1 61 2.4 2.5 Experimental protocol Animal used 61 In-vivo electroporation 61 Collection of 24-hour urine sample 61 Collection of blood sample 62 2.3.2 Urine albumin ELISA 67 2.3.3 Serum biochemistry 67 Serum albumin 67 Serum cholesterol 68 Serum creatinine 68 2.3.4 Serum IgE ELISA 68 2.3.5 Serum IL-13 ELISA 69 2.3.6 Sacrificing of animals 70 Pathological examination 70 2.4.1 Histology 70 2.4.2 Immunofluorescence study 70 2.4.3 Electron microscopy 71 Glomerular gene expression study 73 2.5.1 Isolation of glomeruli by graded sieving technique 73 2.5.2 Extraction of total RNA from isolated glomeruli by TRIzol® reagent 75 2.5.3 Plasmid standards for real-time PCR 75 v Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 2.5.4 Reverse transcription and real-time PCR 76 2.6 Glomerular protein expression study 80 2.7 Flow cytometric analysis of splenic mononuclear cells 83 2.8 Hepatic gene expression study by real-time PCR using hybridization probes 86 2.9 Gene expression study on hepatocyte cell culture incubated with recombinant IL-13 89 2.9.1 Incubation of hepatocyte cell line with recombinant IL-13 89 2.9.2 Gene expression study 89 2.10 Statistical analysis CHAPTER OVEREXPRESSION OF INTERLEUKIN-13 AND DEVELOPMENT OF MINIMAL CHANGE NEPHROTIC SYNDROME 93 94 3.1 Introduction 94 3.2 Results 97 3.2.1 Body weight 97 3.2.2 24-hour urine albumin excretion 98 3.2.3 Serum biochemistry 99 3.2.4 Light microscopy & transmission electron microscopy 102 3.2.5 Flow cytometric analysis of splenic mononuclear cells 104 3.3 Discussion 106 3.4 Future directions 112 CHAPTER 4.1 MOLECULAR MECHANISM OF PROTEINURIA IN MCNS INDUCED BY INTERLEUKIN-13 Introduction 113 113 vi Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 4.2 4.3 Results 116 4.2.1 Quantitation of real-time PCR 116 4.2.2 Glomerular expression of IL-13 receptors and STAT6 118 4.2.3 Glomerular expression of nephrin, podocin and dystroglycan 120 Discussion 124 CHAPTER ASSOCIATION OF B7-1 WITH PROTEINURIA IN MCNS INDUCED BY INTERLEUKIN-13 130 5.1 Introduction 130 5.2 Results 132 5.2.1 Glomerular expression of B7-1 132 5.2.2 Correlation between serum IL-13 levels and glomerular gene expression of B7-1 135 Discussion 136 5.3 CHAPTER 6.1 6.2 EFFECTS OF INTERLEUKIN-13 ON CHOLESTEROL METABOLIC PATHWAY 140 Introduction 140 6.1.1 140 Nephrotic syndrome and hypercholesterolemia 6.1.2 Cholesterol metabolism 141 6.1.3 147 Hepatic enzymes and hypercholesterolemia in nephrotic syndrome 6.1.4 Aim of chapter 148 Results 150 6.2.1 Biochemical data 150 6.2.2 Effect of IL-13 on weight of liver 152 6.2.3 Hepatic gene expression of enzymes and proteins associated with cholesterol metabolism in IL-13 overexpression rat model 153 vii Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 6.3 6.2.4 Hepatic gene expression of IL-13 receptor subunits and STAT6 in IL-13 overexpression rat model 155 6.2.5 157 Correlation studies 6.2.6 Gene expression of enzymes and proteins associated with cholesterol metabolism in mouse hepatocyte cell line incubated with recombinant IL-13 161 6.2.7 Gene expression of IL-13 receptor subunits and STAT6 in mouse hepatocyte cell line incubated with recombinant IL-13 163 Discussion 165 CHAPTER CONCLUSION 171 BIBLIOGRAPHY 174 APPENDICES 217 viii Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Reagent preparation: Wash buffer 1x PBS 990 ml FBS (1%) 10 ml Sodium azide, NaN3 (0.1%) 1g RBC lysis buffer 155 mM NH4Cl 10 mM KHCO3 (omitted) 0.1 mM EDTA NH4Cl (MW = 53.5) M = 53.5 g in litre 155 mM = 8.3 g in litre = 4.15 g in 500 ml EDTA (MW = 372.24) M = 372.24 g in litre 0.1 mM = 0.037 g in litre = 0.0186 g in 500 ml 0.1 M EDTA = 1.86 g in 50 ml ddH2O To prepare 500 ml of working RBC lysis buffer: ddH2O 500 ml 0.1 M EDTA 0.5 ml NH4Cl 4.15 g 273 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 2.27 Harvesting of hepatocytes for gene expression study Procedures: 1. At the end of incubation, remove the culture medium. 2. Rinse with ml of sterile 1x PBS. 3. Incubate with ml of 1x trypsin-EDTA (JRH, cat.#59418) in 1x PBS (sterile) for 10 minutes at 37 oC. 4. Neutralize trypsin with ml of culture medium (10% FBS in DMEM). 5. Transfer the cell suspension into a 10 ml centrifuge tube. 6. Centrifuge at 2000 rpm for minutes, decant the supernatant. 7. Disperse cells, resuspend in 10 ml of 1x HBSS. 8. Centrifuge at 2000 rpm for minutes, decant the supernatant. 9. Disperse cells, add ml of TRIzol® reagent, vortex briefly, stand at room temperature for 10 minutes. Keep at -80oC. 274 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 2.28 Preparation of common reagents 1x Hank’s balanced salt solution (HBSS) Sterile ddH2O 450 ml 10x HBSS (Gibco BRL, cat.#14065-056) 50 ml 7.5% NaHCO3 solution ml Before adding of 7.5% NaHCO3 solution, pH of 1x HBSS = 6.52 After adding ml of 7.5% NaHCO3 solution, pH of 1x HBSS = 7.4-7.5 7.5% NaHCO3 solution (MW = 84): NaHCO3 7.5 g Sterile ddH20 100 ml Filtered by 0.22 m filter using a syringe. 10x Phosphate buffered saline (PBS) NaCl 80 g KCl 2g Na2HPO4 14.4 g KH2PO4 2.4 g Dissolve in 800 ml of ddH2O, adjust pH to 7.4, adjust volume to litre with ddH2O 275 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents 1x PBS 10x PBS stock solution 100 ml ddH2O 900 ml Autoclave if necessary. (When prepared with ddH2O in paediatrics lab, pH of 10x solution = 6.91, after dilution with ddH2O to 1x PBS, pH = 7.45. Therefore, there is no need to adjust pH after preparing the 10x solution.) 10x Tris buffered saline (TBS), 0.05 M, pH 7.6 Tris base 60.6 g NaCl 87.7 g Concentrated HCl 37 ml Top up to litre with ddH2O. 1x TBS, 0.05 M, pH 7.6 10x TBS stock solution 100 ml ddH2O 900 ml 276 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.1 Volume (ml) of 24-hour urine collected at regular intervals Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Day 16 20 30 14 15 16 24 16 24 22 22 20 15 15 36 24 35 14 14 22 20 30 17 12 16 24 12 24 13 26 15 16 23 16 18 18 11.5 6.5 15 10 23 16 18 14 10.5 19 7.5 16 11 Day 14 17 19 26 16 21 34 17 16 31 20 24 NA NA NA NA NA NA 14 18 14 19 42 24 26 66 42 37 19 11 18 24 11 21 34 15 45 19 21 36 15 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Day 28 30 29 26 45 12 8.5 10 12 18 18 27 7.5 13.5 34 43 16 38 17 28 34 44 27 20 65 28 24 16 24 10 40 35 10 28 24 30 49 16 12 31 26 26 14 14 21 13 16 32 13 26 17 NA 22 18 40 12 26 16 Day 42 33 29 27 34 17 15 19 13 21 24 22 17 12 45 36 16 20 14 38 57 20 28 30 20 30 21 21 25 21 21 28 30 20 19 21 NA 17 27 17 24 34 16 20 26 14 20 20 64 30 24 24 16 32 26 37 38 62 36 Day 56 22 30 29 30 24 35 17 17 29 16 23 17 18 26 48 26 30 13 10 35 18 52 18 35 33 29 58 27 25 20 35 48 10 33 23 NA 26 42 NA 28 29 28 10 17 24 15 32 11 22 17 NA 44 22 33 18 40 20 End 26 20 34 19 13 19 23 23 25 15 25 33 20 29 66 38 35 10 10 33 24 52 28 18 30 46 42 28 28 20 13 28 18 18 19 30 28 22 17 34 30 30 32 12 16 29 25 15 20 10 16 27 22 30 27 41 15 277 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.2 Concentration of albumin ( g/ml) in urine measured by ELISA Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Day 11.4 16.6 6.5 28.5 11.4 11.6 24.5 26.9 10.9 14.1 11.6 14.9 11.4 9.7 11.0 29.7 16.7 14.5 12.1 8.0 12.6 8.8 12.0 13.2 9.4 8.9 8.6 12.5 54.6 44.1 16.0 20.9 23.8 14.2 12.0 16.6 20.6 69.8 13.6 18.4 12.4 8.6 14.2 13.8 21.3 30.9 23.6 12.7 16.0 9.5 14.1 31.9 17.3 23.9 13.4 21.7 45.6 32.6 Day 14 12.5 14.4 8.0 10.1 8.9 7.7 12.8 10.5 12.1 10.8 12.3 NA NA NA NA NA NA 11.1 20.9 13.2 13.6 7.6 11.8 9.6 8.4 7.8 12.4 14.7 119.0 48.7 10.5 92.0 14.9 92.1 23.8 10.0 13.7 130.8 9.4 46.1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Day 28 8.6 10.5 13.8 7.4 16.5 24.3 12.5 11.8 25.8 13.4 13.3 32.6 10.7 7.1 6.7 14.7 6.2 12.9 17.7 9.3 20.9 7.9 10.8 11.5 11.9 8.8 9.5 17.1 198.7 95.2 9.2 43.9 33.6 19.3 20.7 402.9 10.1 126.0 330.6 24.0 12.5 8.8 13.7 12.3 10.8 62.5 16.0 7.9 51.1 8.7 17.1 NA 10.8 4.5 21.2 17.2 93.8 36.2 Day 42 9.5 9.9 9.2 8.9 14.3 13.6 10.4 12.4 11.5 12.4 13.4 14.2 14.9 11.2 10.9 14.4 13.9 10.3 19.1 1123.6 204.0 9.2 123.8 13.5 358.4 12.7 20.1 26.4 383.4 114.5 19.2 157.8 27.4 38.3 36.0 NA 29.8 306.5 1932.0 159.9 20.8 17.6 44.8 15.6 40.2 151.7 22.5 12.6 37.1 11.4 18.7 13.7 15.0 28.3 42.0 22.0 64.9 104.3 Day 56 13.8 9.0 9.9 9.9 184.5 8.4 13.6 14.8 26.2 15.3 14.8 16.5 15.0 14.7 14.8 15.6 14.9 10.5 393.0 9.5 210.5 26.6 30.3 12.6 104.3 12.2 16.1 43.7 179.6 153.5 25.7 139.4 42.5 24.0 42.5 NA 12.9 117.5 NA 590.0 48.1 15.9 45.5 22.1 54.1 162.8 28.6 17.1 415.7 20.3 25.0 NA 21.5 24.6 54.6 22.0 91.4 38.2 End 15.3 10.9 8.9 10.2 17.8 8.7 18.4 23.0 27.4 23.5 24.0 13.6 9.3 8.4 7.5 8.7 7.9 15.1 233.1 39.3 129.1 181.8 136.0 251.0 253.0 16.8 44.4 107.0 174.7 66.4 119.9 244.5 104.6 107.6 158.5 402.9 86.3 83.2 1932.0 1000.0 35.1 35.4 46.1 24.5 72.4 150.4 343.5 56.1 178.6 17.4 147.8 13.7 104.2 107.4 76.4 19.0 120.2 187.8 278 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.3 24-hour urine albumin excretion ( g/24hr) Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Day 183 332 195 399 171 186 196 161 262 225 278 89 251 213 221 267 251 131 181 289 301 308 169 184 206 178 258 213 437 529 256 501 285 341 156 432 308 558 218 424 199 154 255 159 138 464 236 292 128 152 254 160 242 251 254 162 729 359 Day 14 213 274 208 162 187 262 218 168 375 216 295 NA NA NA NA NA NA 155 376 185 258 319 283 250 554 328 459 279 1309 877 252 1012 313 3131 357 450 260 2747 338 692 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Day 28 258 305 359 333 198 207 125 142 464 241 358 245 144 241 286 235 234 116 301 260 711 348 292 231 776 248 228 274 4770 952 368 1537 336 542 497 12087 495 2016 3967 744 325 228 192 172 226 813 257 251 664 227 291 NA 238 81 849 207 2439 579 Day 42 314 287 248 303 243 204 198 161 242 298 295 241 178 503 392 231 278 144 726 64045 4080 258 3714 270 10751 268 422 659 8051 2405 538 4734 548 728 755 NA 506 8276 32845 3837 706 282 897 406 562 3033 450 808 1113 274 450 219 481 737 1555 834 4025 3755 Day 56 304 270 287 297 4428 294 232 251 761 245 340 280 271 382 710 406 446 137 3930 333 3789 1383 545 442 3440 353 936 1180 4490 3071 901 6693 425 791 978 NA 335 4934 NA 5310 1345 462 1275 221 920 3908 429 547 4572 446 426 NA 945 541 1803 396 3656 765 End 398 218 303 194 231 165 424 529 685 352 600 448 186 242 495 332 275 151 2331 1297 3098 9454 3808 4518 7590 773 1863 2996 4891 1328 1559 6846 1884 1937 3012 12087 2417 1831 32845 34000 1054 1062 1474 294 1159 4361 2405 1404 2680 348 1478 219 2813 2363 2293 512 4927 2818 279 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.4 Serum biochemistry at day Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Serum albumin (g/L) 35.6 40.4 37.2 40.0 39.6 43.2 41.2 39.1 40.5 36.6 41.2 44.1 42.5 NA 41.9 51.7 50.5 40.0 36.4 40.8 40.8 44.0 43.2 37.0 40.8 43.6 42.2 38.4 44.7 41.9 44.0 44.7 42.9 46.7 46.4 45.0 41.5 45.0 41.2 38.4 44.4 45.7 53.3 47.3 47.9 46.0 47.0 45.7 45.1 41.6 48.3 47.0 48.9 49.2 NA 46.3 40.6 42.2 Serum cholesterol (mmol/L) 1.97 1.81 1.14 1.81 1.68 1.71 1.50 2.05 1.63 1.71 1.58 1.76 1.94 NA 1.76 1.58 1.97 1.99 1.74 2.12 1.71 1.71 1.99 1.79 1.40 1.53 1.53 1.58 1.76 1.63 1.97 1.66 1.63 1.58 1.63 1.71 1.63 1.74 1.48 1.66 2.05 1.84 2.02 1.94 1.81 2.15 1.89 1.42 2.10 1.86 1.76 1.42 1.45 1.63 NA 1.71 1.71 1.71 Serum creatinine ( mol/L) 74.3 61.9 91.1 83.1 77.8 68.1 82.2 63.6 69.8 56.6 69.0 73.4 84.9 NA 87.5 77.8 87.5 78.7 84.0 87.5 103.4 84.0 84.0 61.0 76.0 72.5 57.5 74.3 84.9 72.5 74.3 82.2 67.2 54.8 72.5 77.8 71.6 97.2 62.8 76.9 76.9 80.4 83.1 76.9 73.4 88.4 65.4 75.1 79.6 79.6 70.7 87.5 76.9 77.8 NA 84.9 94.6 69.8 280 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.5 Serum biochemistry at day 30 Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Serum albumin (g/L) 40.8 39.6 38.8 49.6 45.2 46.8 NA NA NA NA NA 44.7 43.4 47.2 50.4 50.4 45.0 36.8 32.4 35.2 30.0 41.6 39.6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 42.4 44.7 50.7 47.6 44.4 48.5 46.9 42.4 52.0 53.6 46.6 44.7 48.2 49.8 50.4 49.8 49.8 47.2 Serum cholesterol (mmol/L) 1.71 1.55 1.24 1.40 1.14 1.74 NA NA NA NA NA 1.66 2.07 2.15 1.71 1.27 1.45 4.20 2.95 2.43 3.44 1.48 1.81 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 1.99 2.18 2.87 1.94 2.59 1.94 2.25 2.51 1.97 3.06 1.35 1.58 1.84 2.38 1.76 2.02 1.97 1.84 Serum creatinine ( mol/L) 93.7 91.1 86.6 67.2 76.0 84.0 NA NA NA NA NA 63.6 67.2 59.2 68.1 66.3 60.1 117.6 92.8 84.0 75.1 95.5 91.1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 66.3 70.7 69.8 64.5 63.6 64.5 61.0 70.7 76.9 57.5 69.8 70.7 81.3 68.1 71.6 53.0 76.0 72.5 281 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.6 Serum biochemistry at day 60 Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Serum albumin (g/L) 40.0 44.8 NA 48.4 46.4 47.2 45.2 38.0 44.0 49.6 41.6 41.2 37.6 45.2 NA 44.8 47.2 36.0 35.2 35.2 NA 45.6 43.2 42.0 35.6 45.6 36.0 35.2 45.2 40.8 34.8 34.4 37.2 38.4 40.0 NA 39.6 34.8 NA 32.8 49.6 45.6 45.2 43.2 42.0 43.6 53.6 32.4 40.8 51.6 43.6 33.6 39.2 44.0 49.6 39.2 32.8 45.6 Serum cholesterol (mmol/L) 1.24 1.22 NA 1.24 1.19 1.66 1.45 1.24 1.76 1.42 1.40 1.55 1.81 2.51 NA 1.50 1.55 4.64 3.26 3.42 NA 1.50 1.84 1.97 4.12 2.15 2.87 3.86 1.53 1.76 2.90 3.44 2.25 1.86 2.33 NA 2.33 3.65 NA 3.11 1.81 2.10 2.51 1.68 2.15 1.45 2.18 3.24 2.12 3.11 1.89 1.66 2.02 2.10 2.18 2.31 1.79 2.05 Serum creatinine ( mol/L) 86.6 82.2 NA 84.0 99.0 103.4 84.9 84.0 82.2 103.4 107.8 61.0 51.3 63.6 NA 59.2 54.8 101.7 80.4 84.9 NA 84.0 101.7 84.0 77.8 80.4 108.7 112.3 84.9 84.0 77.8 107.8 89.3 82.2 93.7 NA 86.6 84.0 NA 91.1 54.8 74.3 61.9 71.6 49.5 51.3 68.1 57.5 61.0 63.6 64.5 61.0 48.6 63.6 65.4 61.9 54.8 64.5 282 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.7 Serum biochemistry when the rats were sacrificed Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Serum IL-13 (pg/ml) 0 0 17 0 25 14 12 11 15 12 1578 1073 376 167 32 62 232 33 90 28 21 61 54 336 31 57 37 1753 48 298 657 41 109 646 178 141 78 44 837 198 738 419 24 2015 44 141 138 51 158 130 Serum albumin (g/L) 37.9 41.0 42.4 56.9 38.6 49.7 33.0 32.4 35.7 39.1 32.7 47.7 47.1 46.4 42.4 56.4 46.7 40.0 27.9 35.5 29.3 35.5 44.1 35.4 28.3 40.4 38.8 29.0 36.1 32.7 32.0 29.3 31.0 31.0 33.0 18.9 38.1 33.4 15.5 30.7 51.7 53.4 49.4 50.7 34.1 48.1 43.8 32.8 49.1 58.3 39.1 38.1 39.4 40.4 42.4 53.0 43.8 49.1 Serum cholesterol (mmol/L) 1.81 1.94 1.53 1.63 1.81 1.97 1.61 1.58 1.74 1.55 1.68 1.58 2.07 2.10 1.50 1.55 1.55 5.91 7.80 5.65 5.83 4.07 2.43 1.66 4.82 2.12 3.26 4.30 1.92 2.12 3.76 4.45 2.28 2.49 2.90 8.78 2.62 4.01 12.20 3.60 1.86 2.07 2.90 1.89 2.25 1.68 2.41 3.32 2.28 2.82 2.07 3.37 1.84 2.07 1.66 1.86 1.58 2.25 Serum creatinine mol/L) 89.5 88.2 65.2 67.2 84.9 88.2 70.5 66.2 43.8 57.5 64.0 71.6 84.9 102.5 82.2 76.0 82.2 89.5 69.7 75.7 89.5 91.5 103.3 77.8 53.1 62.6 69.1 70.5 63.3 85.0 80.7 67.6 76.4 61.2 75.7 79.3 72.8 71.3 56.8 66.9 73.4 65.4 78.7 78.7 66.3 78.7 90.2 78.7 79.6 76.0 87.5 74.3 78.7 70.7 89.3 57.5 61.0 92.8 Serum IgE (ng/ml) 107.7 74.4 14.8 3.3 2.9 10.4 35.0 4.1 57.3 18.1 57.0 8.7 4.4 10.7 2.0 7.1 11.6 46.1 22.6 3.9 100.0 41.7 5.9 101.7 5.7 100.0 7.4 72.5 7.2 31.1 14.8 29.3 9.5 100.0 25.2 57.8 12.2 37.8 32.1 8.4 20.1 5.7 9.7 6.9 37.0 37.9 9.6 3.1 10.9 37.5 10.4 2.4 97.0 7.7 2.2 10.1 5.1 40.5 283 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.8 Body weight and weight of kidneys, spleen and liver when sacrificed Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Body wt (gram) 236 266 285 261 273 277 342 299 316 332 320 270 272 241 342 266 298 284 305 259 268 253 292 302 358 321 336 382 312 332 380 336 344 354 387 314 370 353 339 369 305 284 317 262 281 299 313 341 329 258 291 252 319 281 299 261 333 299 Kidney (net wt) 0.66 0.71 0.82 0.76 0.87 0.72 0.99 0.89 0.90 0.87 0.88 0.97 0.93 0.89 1.20 0.81 0.96 0.83 1.13 1.05 0.99 0.72 0.81 0.84 1.09 0.90 0.88 1.08 1.07 0.98 1.12 1.02 0.86 0.96 1.08 1.29 1.00 1.01 1.29 1.22 1.03 0.96 1.20 1.00 0.85 0.96 1.01 1.14 1.25 0.97 0.96 0.94 1.12 1.00 1.19 0.92 1.08 1.19 Spleen (net wt) 0.57 0.57 0.83 0.66 0.58 0.67 0.97 0.74 0.72 0.64 0.81 0.63 0.75 0.68 0.90 0.72 0.74 0.84 2.09 1.16 1.86 0.56 0.93 0.68 0.99 4.07 0.71 1.17 0.72 0.84 1.07 1.31 0.90 0.97 0.95 2.03 1.00 0.97 2.56 0.85 1.08 1.11 0.97 0.80 0.81 1.02 0.97 1.20 1.21 0.84 0.72 0.95 0.83 0.91 0.99 0.78 1.06 0.88 Liver (net wt) 12.5 12.0 15.1 12.4 13.3 14.7 14.1 11.3 10.5 11.3 10.7 8.6 9.3 8.9 12.7 8.3 9.1 18.0 16.5 18.3 19.3 13.2 15.8 11.5 18.6 12.2 12.4 16.8 9.6 13.0 16.7 18.1 12.5 12.5 14.9 9.0 12.3 17.3 10.5 15.2 11.4 10.8 15.7 8.4 11.1 11.7 12.3 15.2 11.4 11.2 8.7 12.0 11.0 12.1 9.9 9.4 13.8 10.3 Kidney (g/100g body wt) 0.280 0.265 0.288 0.291 0.317 0.258 0.289 0.296 0.284 0.261 0.274 0.360 0.340 0.370 0.350 0.310 0.320 0.292 0.371 0.405 0.370 0.283 0.277 0.277 0.305 0.281 0.262 0.283 0.342 0.294 0.295 0.304 0.249 0.272 0.278 0.411 0.269 0.286 0.381 0.329 0.340 0.340 0.380 0.380 0.300 0.320 0.320 0.330 0.380 0.370 0.330 0.370 0.350 0.360 0.400 0.350 0.320 0.400 Spleen (g/100g body wt) 0.242 0.214 0.291 0.253 0.213 0.242 0.284 0.248 0.228 0.193 0.253 0.233 0.276 0.282 0.263 0.271 0.248 0.296 0.685 0.448 0.694 0.221 0.319 0.225 0.277 1.268 0.211 0.306 0.231 0.253 0.282 0.390 0.262 0.274 0.246 0.647 0.270 0.275 0.755 0.230 0.354 0.391 0.306 0.305 0.288 0.341 0.310 0.352 0.368 0.326 0.247 0.377 0.260 0.324 0.331 0.299 0.318 0.294 Liver (g/100g body wt) 5.30 4.51 5.30 4.75 4.87 5.31 4.11 3.78 3.31 3.40 3.33 3.19 3.41 3.69 3.70 3.11 3.05 6.34 5.41 7.07 7.20 5.22 5.41 3.82 5.20 3.80 3.70 4.39 3.06 3.93 4.40 5.40 3.63 3.54 3.85 2.86 3.32 4.91 3.08 4.13 3.75 3.81 4.94 3.19 3.94 3.92 3.92 4.45 3.46 4.35 3.00 4.76 3.45 4.30 3.32 3.61 4.15 3.44 284 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 3.9 Summary of results on flow cytometric study of splenic mononuclear cells Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 % T (CD3) % B (CD45RA) % Macrophage 51.3 55.8 42.8 35.6 44.5 51.7 36.7 40.4 46.5 40.4 46.3 50.9 45.3 46.5 56.4 55.8 49.0 37.3 41.1 22.7 27.4 32.3 42.9 42.3 42.2 40.3 41.4 36.7 35.8 43.7 41.3 32.8 38.5 52.3 46.8 30.7 45.5 38.7 33.2 37.3 47.0 48.3 45.1 43.4 44.1 44.3 43.6 37.1 45.3 45.3 52.5 49.8 52.0 45.5 46.5 28.1 47.4 31.8 34.4 36.4 37.9 35.1 35.7 30.3 31.0 37.2 36.9 38.8 32.8 24.5 25.5 27.7 21.7 26.8 29.4 45.9 43.2 53.0 51.9 44.9 38.3 43.7 41.0 30.4 41.7 44.2 44.7 41.6 42.2 49.8 40.7 32.7 37.3 47.8 34.6 44.4 45.4 40.3 29.3 26.6 29.7 34.7 38.4 29.9 33.3 40.8 34.1 36.2 28.7 27.1 26.0 32.9 23.1 49.2 25.5 26.1 7.3 6.6 15.9 16.8 11.1 7.9 2.3 3.9 4.9 7.0 8.9 8.2 10.0 3.6 8.3 5.4 10.1 7.5 3.7 8.1 8.8 12.7 6.8 4.4 5.8 8.5 13.1 3.2 10.4 3.7 5.5 8.3 6.5 5.8 5.6 5.9 5.5 8.6 4.9 9.5 11.3 8.8 14.4 8.7 6.9 6.9 7.1 6.3 8.7 6.5 8.3 9.0 9.5 9.5 6.4 6.6 14.0 9.2 285 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 4.1 Glomerular gene expression index standardized against β-actin Sample C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 Nephrin 0.110 0.130 0.126 0.148 0.117 0.143 0.040 0.052 0.053 0.064 0.088 0.198 0.189 0.338 0.290 0.370 0.306 0.103 0.011 0.012 0.015 0.112 0.202 0.029 0.005 0.062 0.069 0.031 0.015 0.031 0.016 0.001 0.018 0.018 0.019 0.001 0.025 0.007 0.004 0.014 0.103 0.108 0.082 0.188 0.167 0.212 0.170 0.095 0.102 0.135 0.173 0.285 0.114 0.234 0.219 0.269 0.147 0.147 Podocin 0.225 0.261 0.262 0.202 0.398 0.278 0.052 0.078 0.084 0.076 0.083 0.278 0.299 0.026 0.543 0.554 0.621 0.231 0.018 0.025 0.029 0.199 0.412 0.055 0.010 0.112 0.085 0.033 0.021 0.029 0.017 0.003 0.013 0.015 0.020 0.004 0.025 0.007 0.002 0.016 0.239 0.120 0.110 0.248 0.276 0.290 0.232 0.129 0.155 0.105 0.355 0.367 0.157 0.302 0.279 0.466 0.214 0.496 Dystro 0.0194 0.0243 0.0338 0.0220 0.0407 0.0276 0.0052 0.0060 0.0074 0.0029 0.0105 0.027 0.021 0.010 0.034 0.019 0.028 0.0219 0.0071 0.0093 0.0174 0.0312 0.0350 0.0020 0.0016 0.0016 0.0110 0.0032 0.0021 0.0045 0.0049 0.0013 0.0017 0.0010 0.0046 0.0027 0.0066 0.0028 0.0018 0.0025 0.011 0.012 0.007 0.011 0.013 0.034 0.015 0.011 0.008 0.009 0.018 0.015 0.007 0.011 0.018 0.030 0.010 0.009 B7-1 0.0042 0.0059 0.0067 0.0028 0.0033 0.0025 0.0059 0.0001 0.0030 0.0001 0.0041 0.0139 0.0135 0.002 0.0049 0.0028 0.0037 0.0034 0.0334 0.0093 0.0138 0.0071 0.0046 0.0046 0.0095 0.0097 0.0056 0.0077 0.0001 0.0099 0.0052 0.0108 0.0079 0.0063 0.0077 0.0273 0.0037 0.0067 0.0335 0.0059 0.0122 0.0106 0.0085 0.009 0.0096 0.0189 0.0117 0.0094 0.0352 0.0073 0.0136 0.0101 0.0096 0.0123 0.006 0.0104 0.0109 0.0075 IL-4R 0.0104 0.0113 0.0107 0.0113 0.0105 0.0100 0.0019 0.0018 0.0031 0.0007 0.0045 0.0087 0.0071 0.0048 0.0047 0.0047 0.0039 0.0114 0.0115 0.0129 0.0150 0.0148 0.0130 0.0032 0.0044 0.0061 0.0085 0.0040 0.0052 0.0048 0.0055 0.0046 0.0042 0.0044 0.0071 0.0082 0.0053 0.0054 0.0044 0.0044 0.0151 0.0098 0.0109 0.0118 0.0183 0.0184 0.0147 0.0113 0.0108 0.0101 0.0151 0.0195 0.0127 0.0152 0.0130 0.0145 0.0133 0.0131 IL-13R 0.0075 0.0102 0.0120 0.0061 0.0098 0.0075 0.0035 0.0028 0.0027 0.0017 0.0025 0.0115 0.0078 0.0065 0.0098 0.0097 0.0118 0.0075 0.0078 0.0059 0.0094 0.0097 0.0082 0.0016 0.0021 0.0035 0.0037 0.0022 0.0022 0.0029 0.0029 0.0019 0.0022 0.0020 0.0032 0.0021 0.0030 0.0024 0.0018 0.0023 0.0065 0.0044 0.0049 0.0076 0.0093 0.0096 0.0073 0.0059 0.0060 0.0049 0.0079 0.0099 0.0063 0.0106 0.0095 0.0124 0.0064 0.0070 IL-13R 0.0003 0.0005 0.0004 0.0008 0.0022 0.0009 0.0001 0.1227 0.0232 0.0001 0.0290 0.0001 0.0590 0.0070 0.0058 0.0043 0.0002 0.0023 0.1294 0.0412 0.0089 0.0008 0.0018 0.0210 0.0461 0.0001 0.0457 0.0505 0.0757 0.0189 0.0786 0.1396 0.0865 0.0189 0.0069 1.4493 0.0195 0.0090 0.3250 0.0725 0.0023 0.0061 0.0020 0.0040 0.0260 0.2122 0.0041 0.0129 0.0584 0.0032 0.0325 0.0174 0.0053 0.0160 0.0028 0.0156 0.0047 0.0234 STAT6 0.0166 0.0206 0.0274 0.0128 0.0231 0.0163 0.0125 0.0147 0.0034 0.0323 0.0115 0.0444 0.0245 0.0121 0.0241 0.024 0.0192 0.0151 0.0337 0.0291 0.0406 0.0291 0.0206 0.0073 0.0155 0.0216 0.0211 0.0107 0.01 0.016 0.0191 0.0126 0.0129 0.0091 0.0219 0.0216 0.0179 0.0116 0.0151 0.0141 0.0341 0.0303 0.0208 0.0281 0.0287 0.0429 0.0237 0.0215 0.0251 0.0232 0.0375 0.0398 0.0226 0.0181 0.036 0.0331 0.0207 0.0191 286 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 6.1 Hepatic gene expression index standardized against β-actin SREBP-2 HMG-CoAR CYP7A1 LDLR IL-4Rα IL-13Rα1 IL-13Rα2 STAT6 0.00614 0.0284 0.0297 0.0238 0.0084 0.00198 0.00044 0.00068 C2 0.00094 0.0384 0.0789 0.0204 0.0066 0.00198 0.00012 0.000117 C3 0.00136 0.022 0.2553 0.0489 0.0102 0.00168 0.00028 0.00018 C4 0.00065 0.0505 0.2642 0.0218 0.0074 0.00195 0.00039 0.00007 C7 0.00208 0.0517 0.2 0.0773 0.007 0.0016 0.00039 0.00005 C8 0.00155 0.0642 0.2753 0.059 0.0096 0.003 0.00021 0.000072 C9 0.00114 0.0296 0.0524 0.017 0.0037 0.00161 0.00013 0.000031 C10 0.00067 0.0242 0.1766 0.0211 0.0051 0.00315 0.00023 0.000036 J1 0.00136 0.0524 0.2253 0.0151 0.0107 0.00299 0.00094 0.000048 J2 0.00024 0.0626 0.3869 0.0148 0.0079 0.0025 0.00434 0.000059 J3 0.00086 0.0543 0.3773 0.0175 0.0108 0.00172 0.0039 0.000049 J4 0.00082 0.0692 0.4679 0.0179 0.0142 0.00195 0.00397 0.000153 J5 0.00077 0.0588 0.1093 0.0215 0.0142 0.00274 0.00205 0.000022 J8 0.00186 0.0552 0.2723 0.02 0.011 0.00319 0.0018 0.000064 J11 0.00103 0.0597 0.6084 0.0564 0.0193 0.00725 0.0019 0.000139 J15 0.00077 0.0408 0.3118 0.0244 0.0079 0.00425 0.00347 0.000161 J19 0.00199 0.1027 0.8432 0.0197 0.0095 0.00376 0.0035 0.000051 J21 0.00109 0.0541 0.3397 0.0301 0.0067 0.004 0.00276 0.000041 J22 0.00127 0.0742 0.2836 0.0121 0.0055 0.00305 0.00178 0.000043 J26 0.00136 0.018 0.0245 0.0407 0.0066 0.00131 0.00327 0.000068 J31 0.00294 0.0275 0.3728 0.1708 0.008 0.00106 0.00482 0.000048 J35 0.00188 0.0586 0.2735 0.0684 0.0079 0.00157 0.00469 0.000129 C1 287 Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Appendix 6.2 Gene expression index of hepatocyte cell line incubated with recombinant IL-13 standardized against β-actin SREBP-2 HMG-CoAR LDLR IL-4Rα IL-13Rα1 IL-13Rα2 STAT6 C1 0.00714 0.00707 0.00606 0.000478 0.000452 0.0000154 0.00278 C2 0.00833 0.00694 0.00680 0.000454 0.000514 0.0000136 0.00294 C3 0.00712 0.00727 0.00621 0.000479 0.000524 0.0000149 0.00286 C4 0.01209 0.00684 0.00796 0.000431 0.000556 0.0000150 0.00366 C5 0.01187 0.00732 0.00776 0.000482 0.000593 0.0000181 0.00384 C6 0.01518 0.00836 0.01099 0.000605 0.000626 0.0000219 0.00477 50ng (1) 0.00921 0.00811 0.00820 0.000536 0.000598 0.0000351 0.00362 50ng (2) 0.00942 0.00788 0.00723 0.000544 0.000601 0.0000319 0.00373 50ng (3) 0.01309 0.00834 0.01068 0.000631 0.000624 0.0000404 0.00426 50ng (4) 0.01318 0.00854 0.00972 0.000692 0.000616 0.0000411 0.00444 50ng (5) 0.01344 0.00977 0.01085 0.000675 0.000682 0.0000420 0.00421 50ng (6) 0.00894 0.00850 0.00865 0.000638 0.000590 0.0000412 0.00372 288 [...]... rat IL -13 mRNA 217 Appendix 2.2 Setup of Polymerase Chain Reaction (PCR) 218 Appendix 2.3 TA Cloning, transformation and amplification of plasmid DNA 220 Appendix 2.4 Gel extraction and purification of DNA 223 Appendix 2.5 Extraction and purification of plasmid DNA using Qiagen QIAprep Spin Miniprep kit 225 Appendix 2.6 Extraction and purification of DNA after cycle sequencing 227 Appendix 2.7 Extraction... leads to phosphorylation of STAT6, a transcription factor that regulate genes’ expression (Kelly-Welch et al, 2003) This explains the striking overlap of IL-4 and IL -13 induced biological activities such as regulation of antibody production and inflammation 1.1.5 Pathological role of IL -13 Apart from its effects on the haematopoietic cells of the immune system, IL -13 also plays a role in a number of. .. IL -13 (Yasunaga et al, 2003; Yoshikawa et al, 2003) and acts to downregulate the effect of IL -13 Both IL-13Rα1 and IL-13Rα2 are members of the class I cytokine receptor family They contain an N-terminal immunoglobulin-like domain followed by a class I cytokine receptor region including a WSXWS motif near the transmembrane domain The human and mouse IL-13Rα1 proteins have a cytoplasmic region capable of. .. in-vivo and in-vitro conditions The gene expression of HMG-CoA reductase, the rate limiting enzyme in the cholesterol synthetic pathway, was found to be upregulated in the liver of the IL -13 transfected rats as well as in hepatocyte cell culture incubated with recombinant IL -13 Chapter 7 is a summary and conclusion on the results of this study The role of IL -13 on the development of MCNS was reexamined based... Upgrade PDF Complete Expanded Features Unlimited Pages Documents between IL -13 and IL-4 proteins (Chomarat & Banchereau, 1998) The IL-4 and IL13 receptors are multimeric and share at least one common chain called IL-4Rα Both cytoplasmic tails of IL-4 and IL -13 receptor subunits associate with tyrosine kinases of the Janus family Activation of JAKs initiates a series of downstream signal transduction... synthesis in a rat model of minimal change- like nephropathy overexpressing IL -13 xxvi Click Here & Upgrade PDF Complete Expanded Features Unlimited Pages Documents Awards 1 Abstract based on the work of IL -13 over-expression rat model, titled “Overexpression of interleukin- 13 induces minimal change- like nephropathy in rats and is associated with increased B7-1 expression in the glomeruli”, was awarded... in detail in the “Appendix” section Chapter 3 is a study on the effect of IL13 on the development of proteinuria and other clinico-pathological features of MCNS Rats overexpressing IL -13 developed varying degrees of proteinuria, hypoalbuminemia and hypercholesterolemia with 17% of the IL -13 transfected rats developing frank nephrotic syndrome No significant pathological changes were found in the glomeruli... which are podocyte-associated proteins important in maintaining the glomerular filtration barrier, were found to be significantly decreased in the IL -13 transfected rats when compared to the control rats This was paralleled by an increase in the glomerular gene and protein expression of IL-4Rα, one of the IL -13 receptor subunits, suggesting that IL -13 may act directly on the podocytes and cause the ix... signal transduction and contain Box 1 and Box 2 motifs The main structural difference between the two IL -13 receptors is that the IL-13Rα1 chain has a longer intracellular domain than IL13Rα2 (Hilton et al, 1996; Caput et al, 1996) IL -13 receptors are expressed on human B cells, basophils, eosinophils, mast cells, endothelial cells, fibroblasts, monocytes, macrophages, respiratory epithelial cells, and... pathological conditions in the body Studies on IL -13 have been focused on its close relationship to asthma and allergic diseases It has also been involved in tissue fibrosis, tumor immunosurveillance, Hodgkin lymphoma and resistance to parasites (Wynn, 2003) IL -13, asthma and allergic diseases Asthma is a complex inflammatory disease of the lung characterized by airway hyperresponsiveness, subepithelial . i ROLE OF INTERLEUKIN-13 ON THE DEVELOPMENT OF MINIMAL CHANGE NEPHROTIC SYNDROME – A NOVEL ANIMAL MODEL LAI KIN WAI (BSc, MMedSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT. Features Unlimited Pages ix SUMMARY The aim of this thesis is to investigate the role of interleukin-13 (IL-13), a Th2 cytokine, in the induction of minimal change nephrotic syndrome (MCNS) using a novel IL-13. results of this study. The role of IL-13 on the development of MCNS was re- examined based on our initial hypothesis and results obtained from our IL-13 overexpression rat model. The similarities and