Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. ASSESSMENT OF THE EFFECT OF BLOOD CONTAMINATION ON THE URINARY PROTEIN TO CREATININE RATIO IN THE DOG. A dissertation presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies In Veterinary Pathology at Massey University, College of Science, Turitea, Palmerston North New Zealand Eloise Katherine Puia Jillings 2007 ABSTRACT The urine protein to creati n i ne ratio (UPCR) is a reliable method to assess the total urinary protein loss in the dog from a single urine sample . Interpretation of the urine protein to creatinine ratio has been d ifficult in the presence of haematuria in the sample and previously the presence of blood in the urine has negated the use or interpretation of the U PCR. In 2 previous studies blood has been added to the urine sample of a single dog to aid interpretation of the UPCR in the presence of blood contamination . I n this study blood contamination of urine samples i n 2 1 dogs was assessed to develop guidel ines for interpretation of the UPCR in the face of haemorrhage. Blood was added to the urine from the same dog to make samples with blood contamination leve ls ranging from 0 to 5%. Uri ne d ipstick ana lysis, microscopic examination and a UPCR was performed on a l l samples. The current recommended cut off level for UPCR for normal dogs is <0.5. Resu lts greater than 1 .0 are considered abnormal , results greater than 2 .0 suggests glomerular disease, and UPCR resu lts between 0 .5 and 1 .0 are questionable . The results of the present study suggest that when urine is visib ly red , haemorrhage may be considered as a differential for a U PCR up to 2 . 0 . The practice of attributing proteinuria in non discoloured (yellow) ur ine samples with microscopic haemorrhage to the blood present should be discont inued, as microscopic haemorrhage that does not result in a visible change in colour of the urine sample from yellow wi l l not substantial ly increase the U PCR. As such , the UPCR level in yel low urine even in the presence of microscopic haematuria can be considered va l id . ACKNOWLEDGEMENTS There are many people who have contributed towards the completion of my research. Firstly, I wou ld l ike to acknowledge my husband Bevan for h i s encouragement t h roughout th is and a l l that I do . To my wonderfu l parents, Peter and Marina, you r continuing support and infl uence throughout m y life has been pivotal in creating who I h a ve become. Arohanu i ki a koutou katoa. I would l ike to thank my superv isor Keith Thompson for h is gu idance and leadersh ip . You are a g reat example both personal ly and p rofessiona l ly to those of us who have t he opportunity to work with you . Many thanks a lso to Richard Squ i res, for sparking my interest in prote inuria, and to Sandra Forsyth for he r col legia l ity and support. I a m especia l ly g rateful to the s taff of New Zealand Veterinary Pathology for a l lowing m e to use the i r equipment, and putting up with me in thei r workspace. Add it ional ly, I would l ike to thank Alasdair Noble for h is statistical assistance, without which I wou ld have been lost . M y gratitude goes to the veterinarians who submitted larger than normal samples for me to assess, and for a l l the samples from vol unteer dogs be long ing to the staff and students of IVABS and the Massey University Veterinary Teaching Hospita l . This research wou ld not have been possible without you al l . I am grateful to the Foundation for Research , Science and Technology for the ir fi n ancial support through the TOapapa POta iao Maori Fel lowsh ip , and particu larly Christine Romanes for her support and patience. Ki a koe te rangatira a Nick Roskruge, he m ih i aroha ki a koe mo to manaakitanga , t iakitanga hoki maku . Tena koe e hoa. The whakatauk i (proverb) below encapsu lates my appreciation for everyone who has contributed in some way to the completion of my research . Ko koutou ki tena, ko ahau ki tenei kTwai o te kete, ka oti te mahi. With you a l l on that, and with me on this h andle of the basket, the work is done. TABLE OF CONTENTS C hapter 1: P roteinuria in the Dog- A Review of Aetiology C l in ical Signs and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.0 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2.0 Renal Anatomy and F unction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 . 1 Renal Vascu lature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 .2 Glomeru lar Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 .3 Tubular Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 .4 The Interstitium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.0 Causes of Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 . 1 Pre-Renal Prote inuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2 Renal prote inuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 . 2 . 1 Functional Renal Prote inuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 .2 .2 Pathological Renal Protei nuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .2 .2 . 1 Tubular Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .2 .2 .2 Interstitial Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .2 .2 .3 Glomerular Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .2 .2 .3 . 1 Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . 1 0 3 .2 .2 .3 .2 Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 3 .3 Post-Renal Prote inuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 4.0 Clin ical Manifestations of Glomerular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 4 . 1 S ignalment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 4 .2 C l in ical Signs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 5.0 Detection and Diagnosis of Prote inuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 5 . 1 Urine Chemistry D ipsticks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 5 .2 Non-Dipstick U rinary Protein Assessment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 5 .2 . 1 Sulphosal icylic Acid Turbid ity (SSA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 5 .2 .2 Trichloroacetic Acid (TCA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 5 .2 .3 Trichloroacetic Acid-Ponceau S (TCA-PS) . . . . . . . . . . . . . . . . . . . . . . . 23 5 .2 .4 Coomassie Bri l l iant Blue (CBB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 5 .2 .5 Benzethonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 5 .3 24 hour urine sample collection for protein assessment . . . . . . . . . . . . . . . 24 5.4 Urinary Protein to Creatin ine Ratio (UPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 5 .4 . 1 UPCR Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 5 .4.2 Interpretation of the Urine Protein to Creatinine Ratio . . . . . . . . 29 5.4.3 Prognostic Use of the U PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 5.4 .4 Monitoring Progress with the UPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 5 .4 .5 Differentiation of Causes of Glomerular D isease with the U PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 5 .5 M icroalbuminuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 5 .6 Renal Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 6.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Chapter 2 : Assessment of the effect of blood contamination on the urinary protein to creatin ine ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 1.0 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46 2.0 I ntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3.0 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4.0 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5.0 D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 6.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Appendix A: Raw data for colour scoring of urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Appendix B : Raw data for u rinary protei n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Appendix C : Raw data for u rinary creatin i ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Appendix D: Raw data for u rinary protein to creatin ine ratio . . . . . . . . . . . . . . . . . . . . 65 Appendix E : Massey university animal eth ics approval . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 LIST OF F IGURES Figure 1.1 Cut surface of a can ine kidney with amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . 1 4 Figure 1.2 Cut surface of a can ine kidney with amyloidosis after exposure to Lugols Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 Figure 2.1 Scatter plot of colour score versus level of blood contamination of the urine sample, with background colouring to match the colour of each urine sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 LIST OF TABLES Table 1.1 Causes of Canine G lomeru lar D isease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Table 1.2 24 hour urinary protein loss in mg/kg for healthy and prote i nuric dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Table 1.3 Trial results for UPCR in healthy and protein uric dogs . . . . . . . . . . . . . . . . . 28 Table 1.4 Recommendations for the normal , equivocal and abnormal range of UPCR in dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Table 2. 1 The number of ind ividuals (n=1 3) that scored each urine sample by colour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Table 2.2 Colour, urine protei n to creatinine ratios, and urinary d ipstick blood results for a l l u rine samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Table 2.3 The number of samples, and percentage of total samples (n=1 5) with urine protein to creatin ine ratios that exceed varying cut-off l imits as the level of blood contamination i ncreases, when the UPCR of the in itial sample is 0 .5 . . . . . . . . . . . . . . . . . . 53