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Urokinase and Urokinase Receptor in the Urinary Tract of the Dog Trina Racquel Bailey Louisiana State University and Agricultural and Mechanical College

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Urokinase and Urokinase Receptor in the Urinary Tract of the Dog Trina Racquel Bailey Louisiana State University and Agricultural and Mechanical College Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2005 Urokinase and urokinase receptor in the urinary tract of the dog Trina Racquel Bailey Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Veterinary Medicine Commons Recommended Citation Bailey, Trina Racquel, "Urokinase and urokinase receptor in the urinary tract of the dog" (2005). LSU Master's Theses. 1457. https://digitalcommons.lsu.edu/gradschool_theses/1457 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. UROKINASE AND UROKINASE RECEPTOR IN THE URINARY TRACT OF THE DOG A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science In The Interdepartmental Program in Veterinary Medical Sciences through the Department of Veterinary Clinical Sciences by Trina Racquel Bailey BSc, Doctor of Veterinary Medicine Atlantic Veterinary College, University of Prince Edward Island, 2000 December 2005 To my husband John, who has been there to help me through it all. To my son Ewan, who makes everything worthwhile. To my family and family in law for all their love, help and support. To Dude, Bailey, Sarah, Scamp, Ellie, Abby, and all the other wonderful animals who have been there for me to love and have allowed me to learn. ii Acknowledgements I would like to acknowledge Dr Inder Seghal for helping me get started, work though the middle and finish the research for my masters program. Thank- you for all you time, support, and expertise. I could not have achieved this without your help. Thank-you to Dr Ramanj Lahiri for your willingness to help a struggling veterinary researcher. I am very grateful for all his advice, suggestions and support. Thank-you to Dr Daniel Paulsen for his expert interpretation of my immunohistochemistry slides and all the hours it took to read them. I appreciate all of his time and help. Thank-you to Kyle Waite for his help and suggestions in finishing the last stages of this project. His Western Blots are the best! Thank-you to Julie Millard for her help and expertise in creating the beautiful slides for immunohistochemistry. Thank-you to Tammy Kessler for her help in the infancy of this project. She isolated uPA in the first samples I collected and set the stage for everything else. She has been a wealth of information and has always been willing to help someone she has never even met. Thank-you to Dr Giselle Hosgood, Dr Loretta Bubenik and Dr Glenn Pettifer, the members of my masters committee. Thank-you to the Louisiana State University School of Veterinary Medicine VCS CORP Funds for financial support. iii Table of Contents Dedication………………………….…………………………………………………….ii Acknowledgments………………………………………………………………....……iii List of Tables..………………………………………………………………………….viii List of Figures …………………………………………………………………….…….ix List of Abbreviations……………………………………………………………………xii Abstract…..……………………………………………………………………………..xiii Chapter 1 Introduction……………………………………………………….…………1 1.1 Urinary Tract Surgery in Animals………..……………………….………...…1 1.2 Factors Contributing to Post-Operative Urinary Tract Hemorrhage……….1 1.3 The Role of the Fibrinolytic System in Urinary Tract Surgery……………...1 1.4 Urokinase and Urokinase Receptor in the Urinary Tract…………………...1 1.5 Clinical Significance in Dogs…………………………………...…………...…2 1.6 Investigational Objectives…………………………………………...…………3 1.7References……………………………………………………………………….3 Chapter 2 Literature Review…………………………………………………………...5 2.1 Introduction………………………………………………………………….5 2.2 Hemostasis………………………………………….………………………5 2.2.1 Vascular Constriction…………………………………………….5 2.2.2 Platelet Plug Formation………………………………………….5 2.2.3 Blood Coagulation and Fibrin Plug Formation………………...7 2.2.4 Clot Lysis…………………………………………………………..7 2.3 The Coagulation System…………………………………………..………7 2.3.1 The Extrinsic Pathway……………………………………………8 2.3.2 The Intrinsic Pathway…………………………………………..10 2.3.3 The Common Pathway…………………………...…………….10 2.4 The Fibrinolytic System...............................................................…....11 2.5 Tissue Plasminogen Activator...................................................…......13 2.6 Urokinase………………………………………………………………......15 2.7 Urokinase Receptor...................................................................…......17 2.8 Plasminogen and Plasmin………..………………………………………19 2.9 Endogenous Inhibitors of Fibrinolysis……………………..…….………21 2.9.1 Inhibitors of Plasminogen and Plasmin………….……………22 2.9.2 Inhibitors of Plasminogen Activators………………….……....23 2.10 Fibrinolysis and the Dog……………………………………………......24 2.11 Urokinase and Urokinase Receptor in the Kidney……………………25 2.12 Urokinase and Urokinase Receptor in the Ureter…………………….26 2.13 Urokinase and Urokinase Receptor in the Bladder…………..………27 2.14 Urokinase and Urokinase Receptor in the Prostate…………….……27 iv 2.15 Urokinase and Urokinase Receptor in the Testicle…………….…….29 2.16 Urokinase and Urokinase Receptor in the Urethra……………......…29 2.17 Urokinase and Urine……………………………………………….……29 2.18 Clinical Importance of the Fibrinolytic System……………….….……30 2.18.1 Coagulopathies and Hypercoagulation…………….………..30 2.18.2 Urokinase and Urokinase Receptor in the Vascular System……………………………………………………………...…..32 2.18.3 Urokinase and Urokinase Receptor in Wound Healing……33 2.19 Summary………………………………………………………………….33 2.20 References…………………………………………………………….....34 Chapter 3 Hypothesis and Objectives……………………………………………….45 3.1 Overall Goals…………………………………………………...………….45 3.2 Hypothesis and Specific Objectives……………………………….…....45 3.2.1 Hypothesis………………………………………………….…....45 3.2.2 Specific Objectives………………………………………..…….46 Chapter 4 Sample Selection and Preparation…………………….………………..47 4.1 Sample Populations…………………………………………….…………47 4.1.1 Sample Population for Urine Collection………………..……..47 4.1.2 Sample Population for Urinary Tract Tissue Collection….….47 4.2 Sampling Technique………………………………………………………48 4.2.1 Urine Collection………………………………………………....48 4.2.2 Urinary Tract Tissue Collection.............................................48 4.3 Sample Preparation……………………………………………………….49 4.3.1 Urine Preparation………………………………...……………..49 4.3.2 Tissue Preparation for Morphologic Study…………..……….50 4.4 References…………………………………………………………………51 Chapter 5 ELISA and Activity Assays for Urokinase in Urine…………………….52 5.1 ELISA Assay Introduction……………………………………………...…52 5.2. ELISA for Urokinase Quantification in Urine………………………......53 5.2.1 Samples……………………………………………………...…..53 5.2.2 Antibodies Purified Enzymes and Buffers……......................54 5.2.3 ELISA Technique………………………………………………..55 5.2.4 Problems…………………………………………………….......56 5.3 Activity Assays Introduction………………………………………………57 5.3.1 Chromogenic Assays………………………………………...…57 5.3.2 Fluorogenic Assays……………………………………………..58 5.4 Fluorogenic Activity Assay for Urokinase Quantification in Urine…....58 5.4.1 Samples………………………………………………………….59 5.4.2 Substrates and Buffers…………………………...…………….59 5.4.3 Fluorogenic Assay Technique……………………...………....59 5.5 Statistical Analysis…………………………………………………….…..61 5.6 Results……………………………………………………………………...62 5.7 Discussion……………………………………………………………........62 5.8 References………………………………………………………...……....72 v Chapter 6 Urokinase and Urokinase Receptor Identification with SDS-PAGE, Western Blot and Immunoprecipitation…………………………………………..….75 6.1 Introduction…………………………………………………………...……75 6.2 SDS PAGE…………………………………………………………………75 6.3 Western Blot…………………………………………………………...…..77 6.4 SDS PAGE and Western Blot for Urokinase…………………..............79 6.4.1 Samples……………………………………………………...…..79 6.4.2 Antibodies, Purified Enzymes and Buffers…………….…..…80 6.4.3 SDS PAGE Procedure……………………………………..…..82 6.4.4 Western Blot Procedure………………………………………..82 6.4.5 Problems………………………………………………….…......83 6.5 Immunoprecipitation………………………………………………………84 6.5.1 Samples………………………………………….………………86 6.5.2 Immunoprecipitation Procedure……………………………….86 6.6 Results………………………………………………………………...……87 6.6.1 Urokinase without Immunoprecipitation…………………...….87 6.6.2 Urokinase Receptor without Immunoprecipitation……...…...88 6.6.3 Urokinase with Immunoprecipitation……………………...…..89 6.6.4 Urokinase Receptor with Immunoprecipitation………………91 6.7 Discussion……………………………………………………………........91 6.8 References………………………………………………………...…...….93 Chapter 7 Localization of Urokinase and Urokinase Receptor Using Immunohistochemistry…………………………………………………………...……95 7.1 Immunohistochemistry Introduction…………………………………..…95 7.2 Immunohistochemistry for Urokinase and Urokinase Receptor Detection…………………………………………………………………….….99 7.2.1 Samples……………………………………………………...…..99 7.2.2 Antibodies…………………………………………………….….99 7.2.3 Immunohistochemistry Technique for Urokinase Detection………………………………………………………………100 7.2.4 Immunohistochemistry Technique for Urokinase Receptor Detection…………………………………………………………..….101 7.3 Problems………………………………………………………………….101 7.4 Results………………………………………………………………….…102 7.4.1 Urokinase and Kidney………………………………………...103 7.4.2 Urokinase Receptor and Kidney………………………….….104 7.4.3 Urokinase and Ureter………………………………………….105 7.4.4 Urokinase Receptor and Ureter………………………..…….105 7.4.5 Urokinase and Urinary Bladder………………………………107 7.4.6 Urokinase Receptor and Urinary Bladder………………..…107 7.4.7
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